We greatly acknowledge Prof. Elisabeth Paus at the Norwegian Radium Hospital (Oslo University Hospital, Oslo, Norway) for providing the ProGRP standard and the anti-ProGRP monoclonal antibody, M18. The publication fee was covered by a grant from Apoteker Harald Conrad Thaulows legat. Trine Gr&#248;nhaug Halvorsen and L&#233;on Reubsaet are partners in the National Network of Advanced Proteomics Infrastructure (NAPI) consortium, which is funded by the Research Council of Norway INFRASTRUKTUR-program (project number: 295910).

Serum from healthy blood donors was used for the preparation of standard solutions. The use of serum from healthy blood donors was performed in strict accordance with Norwegian law. Informed consent was obtained from all subjects. Serum samples were analyzed using methods in accordance with relevant guidelines and regulations. The protocol described is a modified version of the method described in previous work14. An overview of the composition of buffers and solutions and how to prepare them can be found in Supplemental Table S1, while the Table of Materials contains materials, equipment, and reagents used in this protocol.
1. Preparation of antibody-coated magnetic beads
<ol>
	<li>Calculate the amount of antibody necessary to prepare the desired number of beads. Use 1 mg of antibody per 50 mg of magnetic beads. 
	NOTE: Typically, 20 &#181;L of a 20 mg/mL bead suspension is used per sample in subsequent experiments (see step 5.1).</li>
	<li>Calculate the antibody volume necessary to prepare the desired number of beads. 
	NOTE: The antibody volume is dependent on antibody concentration and needs to be calculated for each antibody.</li>
	<li>Transfer the desired volume of antibody solution to a 5 mL low-protein-binding microcentrifuge tube. As an example, if the antibody solution contains 1 mg/mL antibody, use 0.4 mL to prepare 1.0 mL of bead suspension (20 mg beads/mL with 1 mg antibody/50 mg beads). 
	NOTE: The antibodies should be in an amine-free buffer. Use low-protein-binding microcentrifuge tubes for all solutions containing proteins to avoid analyte loss due to adsorption.</li>
	<li>Add a small magnetic stirring bar to the antibody solution and adjust the pH to 2.5 under continuous stirring. Use a pH meter with a micro electrode to measure the pH and adjust the pH by stepwise addition of defined volumes of 1.0 M HCl (start by adding 10 &#181;L portions of 1.0 M HCl and reduce the volume as the pH approaches 2.5). Record the total volume of 1.0 M HCl necessary to adjust the pH to 2.5.</li>
	<li>Remove the micro electrode and incubate the acidified antibody on ice on a magnetic stirrer for 1 h. 
	NOTE: Acid treatment of the antibody is performed to promote the correct orientation of the antibodies on the bead. The concentration of HCl may be reduced to 0.5 M or 0.2 M HCl, depending on the buffer concentration in the antibody solution.</li>
	<li>Neutralize the antibody solution. Use a pH meter with a micro electrode to measure the pH and adjust the pH to 7 by stepwise addition of defined volumes of 1.0 M NaOH (start with a 10 &#181;L portion and reduce the volume as the pH approaches 7). Record the total volume of 1.0 M NaOH added. 
	NOTE: The concentration of NaOH should be the same as the HCl concentration used in step 1.4. NaOH is highly corrosive; use protective equipment, including gloves and appropriate eye protection.</li>
	<li>Calculate the desired volume of tosyl activated magnetic beads to be used. 
	NOTE: Typically, it is recommended to use 20 mg beads/mL when performing small-scale coupling. A minimum of 5 mg of beads should be used.</li>
	<li>Mix the magnetic bead suspension thoroughly on a vortex mixer and withdraw a volume containing the desired amount of bead suspension. As an example, to prepare 1 mL of bead suspension, withdraw a volume containing 20 mg of beads (667 &#181;L if the concentration of beads is 30 mg/mL).</li>
	<li>Place the bead suspension on a magnetic rack for 1 min and remove the supernatant. Wash the beads 2x with equal volume of Type 1 H2O (667 &#181;L if a 30 mg/mL bead solution is used to prepare 1 mL of 20 mg/mL antibody-coated beads). Mix using a vortex mixer after each addition of wash solution and place on the magnetic rack for 1 min prior to removing the supernatant.</li>
	<li>Add the following to the magnetic beads: acid-treated antibody, 0.5 M borate buffer (pH 9.5) (1/5 of total volume as the final concentration should be 0.1 M; this equals 0.2 mL to prepare 1 mL of bead suspension), and coupling buffer for antibody coupling (to prepare 1 mL of antibody-coated magnetic beads, the volume of coupling buffer should equal 1 mL - volume of acid treated antibody - 0.2 mL borate buffer). Mix using a vortex mixer. 
	NOTE: The volume of acid-treated antibody equals the volume of antibody solution (0.4 mL in preparation of 1 mL of bead suspension using a 1 mg/mL antibody solution) + the volume of 1.0 M HCl used to adjust the pH to 2.5 + the volume of 1.0 M NaOH used to adjust the pH to 7). 
	NOTE: The composition of 0.5 M borate buffer (pH 9.5) and coupling buffer for antibody coupling can be found in Supplemental Table S1.</li>
	<li>Rotate at ambient temperature overnight using an end-over-end sample mixer, preferably with reciprocal rotation and vibration.</li>
	<li>Centrifuge for 10 min at 239 &#215; g. Place the tube on the magnet for 2 min and remove the supernatant.</li>
	<li>Wash the beads 2 x 2 h and once overnight by rotating in storage buffer for antibody beads&#160;at ambient temperature using an end-over-end sample mixer. Use the same volume as the total volume in step 1.10. Place the tube on the magnet for 1 min and remove the supernatant between each wash. 
	&#8203;NOTE: The composition of storage buffer for antibody beads can be found in Supplemental Table S1.</li>
	<li>Store in the desired buffer (e.g., the same buffer as in step 1.13) using the desired stock concentration of beads (typically 20 mg/mL). Store in the fridge.</li>
</ol>
2. Preparation of 2 mL of trypsin immobilized beads (20 mg/mL beads)
<ol>
	<li>Add 20 mL of 1 mM HCl to 2 mL of NHS-activated agarose beads to wash the beads.</li>
	<li>Mix and centrifuge at 2,655 &#215; g for 5 min. Remove the supernatant.</li>
	<li>Add 20 mL of 0.1 M phosphate buffer (pH 7.8). Mix using a vortex mixer and centrifuge at 2,655 &#215; g for 5 min. Remove the supernatant. 
	NOTE: The composition of 0.1 M phosphate buffer (pH 7.8) can be found in Supplemental Table S1.</li>
	<li>Add 2 mL of 20 mg/mL trypsin (dissolved in cold coupling buffer for trypsin coupling). 
	NOTE: The composition of coupling buffer for trypsin coupling can be found in Supplemental Table S1. Store the buffer in the fridge.</li>
	<li>Incubate for 25 min at 22 &#176;C and 1,100 rpm using a temperature-controlled mixer. Centrifuge at 2,655 &#215; g for 5 min. Remove the supernatant.</li>
	<li>Add 2 mL of modification buffer for the preparation of trypsin beads and incubate for 20 min at 22 &#176;C and 1,100 rpm using a temperature-controlled mixer. Centrifuge at 2,655 &#215; g for 5 min. Remove the supernatant. 
	NOTE: The composition of the modification buffer for the preparation of trypsin beads can be found in Supplemental Table S1. Store the buffer in the fridge.</li>
	<li>Add 10 mL of blocking buffer for the preparation of trypsin beads and incubate for 10 min at 22 &#176;C and 1,100 rpm using a temperature-controlled mixer. Centrifuge at 2,655 &#215; g for 5 min. Remove the supernatant. 
	NOTE: The composition of blocking buffer for the preparation of trypsin beads can be found in Supplemental Table S1. Store the buffer in the fridge.</li>
	<li>Add 2 mL of storage buffer, mix using a vortex mixer, and store in the fridge until use. 
	&#8203;NOTE: The composition of storage buffer for trypsin beads can be found in Supplemental Table S1. Store the buffer in the fridge.</li>
</ol>
3. DSS/VAMS&#160;sampling and subsequent extraction of dried serum
<ol>
	<li>Prepare standards by spiking the serum with ProGRP (or the protein of interest) at the appropriate level. Keep the spiking volume &#8804; 1% of the total volume. 
	&#8203;NOTE: Here, a stock solution of 295 &#181;g/mL ProGRP in water was used for the preparation of spiked serum standards.</li>
	<li>Mix the standards on a vortex mixer.</li>
	<li>Apply 10 &#181;L of serum (spiked standards) onto DBS cards or allow 10 &#181;L of serum to be collected by VAMS (10 &#181;L) using the manufacturer&#39;s guidelines. For DSS, make sure that the spot is within the dotted circle.</li>
	<li>Allow to air dry at ambient temperature for at least 2 h.</li>
	<li>Cut out the whole spot and transfer it to a 2 mL low-protein-binding microcentrifuge tube or remove the VAMS from the holder and place it in a 2 mL low-protein-binding microcentrifuge tube.</li>
	<li>Add 1,000 &#181;L of 100 mM ammonium bicarbonate (ABC) solution. Extract the dried serum from the spot/VAMS for 1 h at 22 &#176;C using a temperature-controlled mixer at 1,000 rpm.</li>
	<li>Transfer the extract to a new 1.5 mL low-protein-binding microcentrifuge tube for tryptic proteolysis.</li>
</ol>
4. Digestion of DSS/VAMS extracts
<ol>
	<li>Use 30 &#181;L of trypsin beads (as prepared in section 2) per sample. Transfer a volume containing trypsin beads for all samples to a new 1.5 mL low-protein-binding microcentrifuge tube, centrifuge at 2,655 &#215; g for 5 min, and remove the supernatant.</li>
	<li>Wash the trypsin beads 2x with 1 mL of cold 50 mM ABC, before resuspending in the same volume that was pipetted out. Centrifuge at 2,655 &#215; g for 5 min and remove the supernatant between steps.</li>
	<li>Add 30 &#181;L of washed trypsin beads to each DSS/VAMS extract to initiate digestion.</li>
	<li>Incubate for 2 h at 37 &#176;C and 1,000 rpm using a temperature-controlled mixer or similar. Centrifuge at 2,655 &#215; g for 5 min and transfer the supernatant (not beads) to a new 2.0 mL low-protein-binding microcentrifuge tube.</li>
	<li>Add 25 &#181;L of 14 ng/mL internal standard (IS) solution containing the stable isotope-labeled (SIL) peptide ALGNQQPSWDSEDSSNF[K_13C6_15N2] in 100 mM ABC solution.</li>
</ol>
5. Capture of proteotypic ProGRP peptide using antibody-coated magnetic beads
<ol>
	<li>Use 20 &#181;L of antibody coated beads (20 mg/mL as prepared in section 1) per extraction. Transfer all antibody-coated beads to a new 1.5 mL low-protein-binding microcentrifuge tube, place on the magnet for 1 min, and remove the storage buffer.</li>
	<li>Wash the magnetic antibody-coated beads with 1 mL of phosphate-buffered saline (PBS), pH 7.4, containing 0.05% polysorbate 20, and 2 x 1 mL of PBS before resuspending in the same volume that was pipetted out. Mix on a vortex mixer and place on the magnet for 1 min between buffer exchanges. 
	NOTE: PBS contains 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 1.8 mM KH2PO4. It is recommended to prepare the solution at a 10x concentration for increased stability. For the composition of 100 mL of 10x PBS, see Supplemental Table S1. Dilution of the 10x PBS 10x will achieve a pH of ~7.4.</li>
	<li>Add 20 &#181;L of washed magnetic bead suspension to each low-protein-binding microcentrifuge tube containing a digested DSS/VAMS extract and IS. Perform immunoextraction for 1 h using an end-over-end sample mixer at ambient temperature.</li>
	<li>Wash the magnetic beads using the following solutions: 500 &#181;L of PBS with 0.05% (v/v) polysorbate 20, 400 &#181;L of PBS, 300 &#181;L of 10 mM Tris HCl (pH 7.4), and 300 &#181;L of 100 mM ABC.
	<ol>
		<li>After the addition of each wash solution, remove the low-protein-binding microcentrifuge tube with beads and wash solution from the magnet rack and invert carefully until homogeneous. Then, place the low-protein-binding microcentrifuge tube on the magnet for 30 s, invert for 30 s, and place on the magnet for 1 min. Remove the wash solution.</li>
		<li>Spin down the remaining suspension using a microcentrifuge. Place on the magnet for 1 min and remove the remaining wash solution.</li>
	</ol>
	</li>
	<li>Add 15 &#181;L of 2% (v/v) formic acid in Type 1 H2O to each sample and incubate for 5 min at 22 &#176;C and 1,000 rpm using a temperature-controlled mixer, or similar, to elute the captured peptides. Place on the magnet for 1 min and transfer the eluate to a new 1.5 mL low-protein-binding microcentrifuge tube. Repeat once and transfer the second eluate to the same low-protein-binding microcentrifuge tube as the first eluate.</li>
	<li>Add 20 &#181;L of 100 mM ABC to each eluate (total volume of 50 &#181;L).</li>
	<li>Centrifuge (microcentrifuge) and transfer 40 &#181;L of the eluate to micro inserts for high-performance liquid chromatography (HPLC) vials.</li>
</ol>
6. Analysis by LC-MS/MS
<ol>
	<li>Use a micro LC triple quadrupole MS with electrospray ionization for high robustness.</li>
	<li>Prepare the LC-MS/MS system for the analysis by purging the pumps with mobile phase A (20 mM FA in H2O and MeCN, 95:5 v/v) and B (20 mM FA in H2O and MeCN, 5:95 v/v).</li>
	<li>Insert the analytical column (C18, 50 mm x 1 mm inner diameter [ID], 3 &#181;m particles).</li>
	<li>Continue to prepare the instrument for analysis by following the start-up procedures for the specific instrument.</li>
	<li>In the instrument software, set the column oven temperature to 25 &#176;C and the flow rate to 50 &#181;L/min (100% mobile phase A).</li>
	<li>Equilibrate the column with mobile phase A for at least 15 min.</li>
	<li>Place the samples in the autosampler.</li>
	<li>Prepare the instrument method for analysis of the ProGRP-specific, tryptic peptide ALGNQQPSWDSEDSSNFK, and its IS. 
	NOTE: Several method parameters are instrument-specific and must be optimized on the specific instrument used. Details of the method parameters used here are described in steps 6.9 to 6.11.</li>
	<li>For the gradient program in LC, use the following settings: flow rate: 50 &#181;L/min; from time 0.0 to 3.0 min: 100% mobile phase A; from time 3.0 to 18.0 min: run a linear gradient from 0% to 50% mobile phase B; from time 18.0 to 18.1 min: increase mobile phase B from 50% to 100%; from time 18.1 to 20.0 min: 100% mobile phase B; from time 20.0 to 20.1 min: decrease mobile phase B from 100% to 0%; from time 20.1 to 30 min: 100% mobile phase A to re-equilibrate the column (use at least 10 column volumes).</li>
	<li>For MS/MS settings, run MS/MS in positive mode using instrument settings that ensure efficient ionization. To follow this protocol, use a spray voltage of +4,000 V, a heated capillary temperature of 270 &#176;C, nitrogen as sheet gas (5 arbitrary units), and argon (2 mTorr) for collision-induced dissociation (CID) fragmentation. If the instrument allows, direct the LC flow to waste the first 2 min (0-2 min) and the last 1 min (29-30 min) of the run, instead of into the MS.</li>
	<li>Use selected reaction monitoring (SRM) transitions for (A) the signature peptide (ALGNQQPSWDSEDSSNFK): 1,005.45&#8594;913.3, 1,005.45&#8594;1,028.3, and 1,005.45&#8594;1,398.5, and (B) the IS SIL peptide (ALGNQQPSWDSEDSSNF[K_13C6_15N2]): 1,009.45&#8594;921.3, 1,009.45&#8594;1,036.3, and 1,009.45&#8594;1,406.5. Use optimized collision energy (35 V in this protocol) for all monitored transitions.</li>
	<li>Prepare a sequence containing the samples to be run using the software available for your LC-MS/MS system. Set the injection volume to 10 &#181;L. 
	NOTE: Make sure to add blank samples and quality control samples as needed.</li>
	<li>Press &#34;Run sequence&#34; in the instrument software to start the sequence.</li>
	<li>Monitor the peak area of the affinity-captured, ProGRP-specific, tryptic peptide ALGNQQPSWDSEDSSNFK, and its IS SIL peptide. 
	NOTE: Selection and confirmation of the signature peptide, ALGNQQPSWDSEDSSNFK, is described elsewhere14.</li>
</ol>

<ol>
	<li>Bang, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fresenius.">Fresenius.</a> Zeitschrift f&#252;r analytische Chemie. 52 (7), 521-523 (1913).</li><li>Guthrie, R., Susi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+phenylalanine+method+for+detecting+phenylketonuria+in+large+populations+of+newborn+infants.">A simple phenylalanine method for detecting phenylketonuria in large populations of newborn infants.</a> Pediatrics. 32 (3), 338-343 (1963).</li><li>Laurell, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+screening+test+for+&#945;1-antitrypsin+deficiency.">A screening test for &#945;1-antitrypsin deficiency.</a> Scandinavian Journal of Clinical and Laboratory Investigation. 29 (3), 247-248 (1972).</li><li>Thielmann, K., Aquino, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+blood+samples+dried+and+stored+on+filter+paper+as+substrate+for+the+electrophoretic+separation+on+hemoglobin+S+from+hemoglobin+A.+A+screening+procedure.">Whole blood samples dried and stored on filter paper as substrate for the electrophoretic separation on hemoglobin S from hemoglobin A. A screening procedure.</a> Clinica Chimica Acta. 35 (1), 237-238 (1971).</li><li>Wada, Y., Fujita, T., Kidoguchi, K., Hayashi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+hemoglobin+variants+in+80,000+Japanese+neonates:+High+prevalence+of+Hb+F+Yamaguchi+(A&#947;T+80+Asp&#8594;Asn).">Fetal hemoglobin variants in 80,000 Japanese neonates: High prevalence of Hb F Yamaguchi (A&#947;T 80 Asp&#8594;Asn).</a> Human Genetics. 72 (3), 196-202 (1986).</li><li>Halvorsen, T. G., McKitterick, N., Kish, M., Reubsaet, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+capture+in+bottom-up+protein+analysis-overview+of+current+status+of+proteolytic+peptide+capture+using+antibodies+and+molecularly+imprinted+polymers.">Affinity capture in bottom-up protein analysis-overview of current status of proteolytic peptide capture using antibodies and molecularly imprinted polymers.</a> Analytica Chimica Acta. 1182, 338714 (2021).</li><li>Halvorsen, T. G., Reubsaet, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+based+affinity+capture+LC-MS/MS+in+quantitative+determination+of+proteins+in+biological+matrices.">Antibody based affinity capture LC-MS/MS in quantitative determination of proteins in biological matrices.</a> TrAC Trends in Analytical Chemistry. 95, 132-139 (2017).</li><li>Neubert, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+biomarker+quantification+by+immunoaffinity+liquid+chromatography-tandem+mass+spectrometry:+Current+state+and+future+vision.">Protein biomarker quantification by immunoaffinity liquid chromatography-tandem mass spectrometry: Current state and future vision.</a> Clinical Chemistry. 66 (2), 282-301 (2020).</li><li>Pan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry+based+targeted+protein+quantification:+methods+and+applications.">Mass spectrometry based targeted protein quantification: methods and applications.</a> Journal of Proteome Research. 8 (2), 787-797 (2008).</li><li>Enderle, Y., Foerster, K., Burhenne, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+feasibility+of+dried+blood+spots:+Analytics,+validation,+and+applications.">Clinical feasibility of dried blood spots: Analytics, validation, and applications.</a> Journal of Pharmaceutical and Biomedical Analysis. 130, 231-243 (2016).</li><li>Londhe, V., Rajadhyaksha, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opportunities+and+obstacles+for+microsampling+techniques+in+bioanalysis:+Special+focus+on+DBS+and+VAMS.">Opportunities and obstacles for microsampling techniques in bioanalysis: Special focus on DBS and VAMS.</a> Journal of Pharmaceutical and Biomedical Analysis. 182, 113102 (2020).</li><li>Denniff, P., Spooner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+hematocrit+on+assay+bias+when+using+dbs+samples+for+the+quantitative+bioanalysis+of+drugs.">The effect of hematocrit on assay bias when using dbs samples for the quantitative bioanalysis of drugs.</a> Bioanalysis. 2 (8), 1385-1395 (2010).</li><li>Denniff, P., Spooner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volumetric+absorptive+microsampling:+A+dried+sample+collection+technique+for+quantitative+bioanalysis.">Volumetric absorptive microsampling: A dried sample collection technique for quantitative bioanalysis.</a> Analytical Chemistry. 86 (16), 8489-8495 (2014).</li><li>Levern&#230;s, M. C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocapture+sample+clean-up+in+determination+of+low+abundant+protein+biomarkers-a+feasibility+study+of+peptide+capture+by+anti-protein+antibodies.">Immunocapture sample clean-up in determination of low abundant protein biomarkers-a feasibility study of peptide capture by anti-protein antibodies.</a> RSC Advances. 9 (60), 34902-34911 (2019).</li><li>Conradie, J. D., Govender, M., Visser, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elisa+solid+phase:+Partial+denaturation+of+coating+antibody+yields+a+more+efficient+solid+phase.">Elisa solid phase: Partial denaturation of coating antibody yields a more efficient solid phase.</a> Journal of Immunological Methods. 59 (3), 289-299 (1983).</li></ol>

The authors have no conflicts of interest to disclose.

The described protocol contains information about how to conduct several important steps in the analysis of low-abundance biomarkers from dried microsamples (DSS and VAMS), including the preparation of trypsin beads and antibody-coated magnetic beads. Based on previous experience, we always treat the antibody with acid prior to bead immobilization to improve the orientation of the antibodies15. 
One of the critical steps in this procedure is the selection of the most suitable microsampling format. First, one must consider if the analyte in question can be determined from whole blood, or if the concentration is influenced by the blood cells and has to be determined in serum or plasma (as for the model analyte, ProGRP).
Both paper- and polymer-based approaches have advantages and limitations; for ProGRP, VAMS provides a clear advantage with respect to analyte recovery after extraction from the sampler. This can, however, probably be optimized using a different extraction solution for the DSS samples. Nevertheless, this potential interaction between the analyte and the sampling material is important to take into account as it might result in increased analytical variation and higher detection limits. As the IS used is an SIL peptide and first added after the digestion, IS corrects for the steps following digestion (e.g., affinity extraction and LC-MS/MS analysis). IS correction is not possible for extraction from DSS/VAMS and the digestion step.
Two types of beads are used in the procedure: trypsin beads for digestion after extraction of the serum sample from the sampler, and antibody-coated magnetic beads for capture of the proteotypic peptide after digestion. A major reason for using trypsin beads, in addition to speeding up the digestion, is to minimize residual trypsin activity in the sample during affinity capture. This is important to avoid tryptic proteolysis of the mAb during affinity capture. 
Agarose beads were used for preparation of the trypsin beads, while magnetic beads were used for preparation of the antibody-coated beads. Agarose beads are less expensive than magnetic beads but have a limitation that separation of the beads from the solution requires centrifugation. This makes the separation of beads and supernatant less efficient than when using magnetic beads. In addition, automation of the workflow is difficult using agarose beads. However, magnetic NHS-activated beads are available and can be used for a more streamlined and automatable sample preparation workflow. 
Microsampling is an important trend in the bioanalysis of both drugs and biomarkers. One challenge with the current approach is the limited amount of sample volume (10 &#181;L), which may be of particular importance in the determination of very-low-abundance analytes such as ProGRP (pg/mL-low ng/mL level). However, this challenge may be circumvented using state-of-the-art analytical equipment. For these low-abundance analytes, the choice of sample preparation is crucial, and selective sample cleanup through antibody-based affinity capture is most often needed. As peptide capture has been shown to provide cleaner extracts and lower detection limits than protein capture (using the same antibody)14, the present method focuses on this approach in combination with microsampling. Another advantage of the peptide capture approach is that the IS SIL peptide also corrects for the affinity capture step. 
In this work, an antibody targeting a protein was used for peptide capture. This is an advantage as the availability of off-the-shelf antibodies targeting proteins are higher than off-the-shelf antibodies targeting proteotypic peptides. However, for an anti-protein antibody to efficiently capture a proteotypic peptide, the epitope needs to be intact after protein digestion. In addition, for many antibodies, the exact epitope is not known, making the search for an anti-protein antibody tedious. This limits the number of available anti-protein antibodies applicable for peptide capture. The described procedure is demonstrated using serum as the matrix and ProGRP as the target analyte. The procedure is intended to be applicable to other matrixes and other target analytes. Instead of using a commercially available anti-protein antibody for the affinity capture of the proteotypic peptide, it is possible to also use custom-made, anti-peptide antibodies. The cleanup efficiency of peptide capture compared to protein capture is illustrated in Figure 5. By exchanging the agarose beads used for the preparation of trypsin beads with magnetic beads, the procedure should also be compatible with robotic sample preparation work stations on the market.

Microsampling has been around for more than 100 years since Ivar Bang described glucose monitoring from DBSs in 19131. After Guthrie and Susi introduced DBSs in 1963 for the determination of phenylalanine in newborns2, the technique has become increasingly widespread. The first reports of DBSs for the sampling and storage of proteins were made in the early 1970s3,4, and a decade later, in the 1980s, we found the first report of mass spectrometry (MS) for the determination of proteins from DBSs5. Despite this early introduction, it was not until after the turn of the century that MS determination of proteins from DBSs and other microsampling techniques became more widespread.
In a clinical context, it is of interest to determine proteins in the diagnosis and follow-up of diseases, as well as for treatment monitoring and doping purposes. This targeted determination of protein analytes by MS from small amounts of dried samples is still challenging, and often requires extensive sample preparation prior to analysis.
Targeted quantitative determination of proteins by MS is commonly performed by applying the bottom-up approach, digesting the proteins to peptides prior to analysis. This procedure produces a myriad of peptides, which makes direct analysis of the digested biological sample challenging. A way of circumventing this is to apply a selective affinity cleanup step upfront of MS analysis either before or after the digestion6,7,8. In this way, the protein of interest (or its proteotypic peptide, if the affinity capture step is performed after the digestion) is selectively isolated from the sample matrix prior to analysis, providing lower detection limits9.
Microsampling using DBS cards has certain advantages compared to conventional blood samples, including low sample volume, less invasive sampling, and increased storage stability. However, the sample matrix is different and can introduce other challenges in the analysis (e.g., dried vs. liquid sample matrix and capillary blood vs. serum or plasma)10,11. Another challenge that is observed with DBSs is the so-called hematocrit effect, where the blood hematocrit affects the sample volume further processed for analysis, and hence introduces interindividual variability in the analysis12. Newer microsampling units, such as VAMS introduced in 201413, address this issue by collecting a fixed volume of blood instead of a blood drop.
This protocol describes a setup for the analysis of low-abundance biomarkers from dried microsamples. After elution, the dried sample is digested and, subsequently, the proteotypic peptide is isolated by peptide affinity capture. The model analyte is the SCLC biomarker ProGRP. As ProGRP cannot be determined reliably from whole blood, serum was used as the sample matrix. Representative results from both DSSs and serum samples collected using VAMS are shown.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetic acid N-hydroxysuccinimide ester</td>
			<td>Carbosynt (Staad, Switzerland)</td>
			<td>FA33719</td>
			<td>Store in freezer below -20 &#176;C</td>
		</tr>
		<tr>
			<td>ALGNQQPSWDSEDSSNF[K_13C6 
			_15N2] (&#8805; 95%)</td>
			<td>Innovagen (Lund, Sweden)</td>
			<td>Not applicable</td>
			<td>Store in freezer below -20 &#176;C</td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate BioUltra (&#8805; 99.5% )</td>
			<td>Sigma Aldrich (St. Louis, MO, USA)</td>
			<td>09830-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Aquasil C18 column, 3 &#181;m, 50 mm x 1 mm</td>
			<td>Thermo scientific (Waltham, MA, USA)</td>
			<td>77503-051030</td>
			<td>Analytical column compatible with 100% aqueous mobile phase</td>
		</tr>
		<tr>
			<td>Benzamidine (&#8805; 95.0% )</td>
			<td>Sigma Aldrich (St. Louis, MO, USA)</td>
			<td>12072</td>
			<td>Store in fridge at 2-6 &#176;C</td>
		</tr>
		<tr>
			<td>Calsium chloride dihydrate&#160; (&#8805; 99% )</td>
			<td>Sigma Aldrich (St. Louis, MO, USA)</td>
			<td>223506-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5804</td>
			<td>Eppendorf (Hamburg, Tyskland)</td>
			<td>5804000010</td>
			<td></td>
		</tr>
		<tr>
			<td>Cloned ProGRP isoform 1</td>
			<td>Radium hospital, Oslo University Hospital (Oslo, Norway)</td>
			<td>Not applicable</td>
			<td>Store in freezer below -20 &#176;C</td>
		</tr>
		<tr>
			<td>Disodium hydrogenphosphate dihydrate (pro analysis)&#160;</td>
			<td>Sigma Aldrich (St. Louis, MO, USA)</td>
			<td>30435-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Disodium hydrogenphosphate dodecahydrate (pro analysis)&#160;</td>
			<td>Merck (Darmstadt, Tyskland)</td>
			<td>1.06579.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads M-280 tosylactivated 10 mL</td>
			<td>Invitrogen (Carlsbad, USA)</td>
			<td>14204</td>
			<td>Store in fridge at 2-6 &#176;C</td>
		</tr>
		<tr>
			<td>DynaMag-2</td>
			<td>Invitrogen (Carlsbad, USA)</td>
			<td>123-21D</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanolamine (pro analysis, &#8805; 99%)</td>
			<td>Sigma Aldrich (St. Louis, MO, USA)</td>
			<td>#02400</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid (&#8805; 99% ) for LC-MS</td>
			<td>VWR International (Radnor, PA, USA)</td>
			<td>84865.260</td>
			<td></td>
		</tr>
		<tr>
			<td>FTA DMPK-C cellulose card</td>
			<td>Whatman (Kent, UK)</td>
			<td>WB129243</td>
			<td>DBS card</td>
		</tr>
		<tr>
			<td>HPLC vials, clear glass, 1.5 mL, 32 x 11.6 mm, Clean Pack</td>
			<td>Nerliens Meszansky (Oslo, Norge)</td>
			<td>LPP 11 09 0519</td>
			<td></td>
		</tr>
		<tr>
			<td>Hulamixer sample mixer</td>
			<td>Invitrogen (Carlsbad, USA)</td>
			<td>101561503016</td>
			<td>Sample mixer with end-over-end mixing and reciprocal rotation and vibration</td>
		</tr>
		<tr>
			<td>Human serum from healty blood donors</td>
			<td>Bloodbank, Ullev&#229;l, Oslo University Hospital (Oslo, Norway)</td>
			<td>Not applicable</td>
			<td>Store in freezer below -20 &#176;C</td>
		</tr>
		<tr>
			<td>Hydrochloric acid fuming 37% (Emsure for analysis)</td>
			<td>Merck (Darmstadt, Tyskland)</td>
			<td>1.00317.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>LC-MS/MS system: Ultimate 3000 system (Autosampler, WPS-3000TRS; Micropump, LPG-3400M; Flow manager, FLM-3300, MIC, 1X2P-10P) and TSQ Quantum access. Controlled by Xcalibur 2.2 SP1.48</td>
			<td>Thermo scientific (Waltham, MA, USA)</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>LiChrosolv Acetonitrile hypergrade for LC-MS</td>
			<td>Merck (Darmstadt, Tyskland)</td>
			<td>1.00029.2500</td>
			<td></td>
		</tr>
		<tr>
			<td>LL Biotrode, Combined glass electrode</td>
			<td>Metrohm (Herisau, Sveits)</td>
			<td>6.0224.100</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stirrer, Type M10</td>
			<td>Franz Morat KG (Eisenbach, Germany)</td>
			<td>10236</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro inserts, glass (31 x 6 mm, 0.1 mL)</td>
			<td>VWR International (Radnor, PA, USA)</td>
			<td>548-0006</td>
			<td></td>
		</tr>
		<tr>
			<td>MilliQ integral 3 with Q-POD</td>
			<td>Merck Millipore (Molsheim, France)</td>
			<td>ZRXQ003T0</td>
			<td>For production of Type 1 water</td>
		</tr>
		<tr>
			<td>monoclonal antibody M18</td>
			<td>Radium hospital, Oslo University Hospital (Oslo, Norway)</td>
			<td>Not applicable</td>
			<td>Store in fridge at 2-6 &#176;C</td>
		</tr>
		<tr>
			<td>Neoteryx Mitra microsampler (10 &#956;L) 4 sampler Clamshell</td>
			<td>Fisher Scientific (Waltham, MA, USA)</td>
			<td>NC1382947</td>
			<td></td>
		</tr>
		<tr>
			<td>NHS-activated sepharose beads 4 fast flow</td>
			<td>Sigma Aldrich (St. Louis, MO, USA)</td>
			<td>GE17-0906-01</td>
			<td>Agarose beads, store in fridge at 2-6 &#176;C</td>
		</tr>
		<tr>
			<td>Optifit, Refill pipet tips, 10 mL</td>
			<td>Sartorius Biohit (Helsinki, Finland)</td>
			<td>613-2911</td>
			<td></td>
		</tr>
		<tr>
			<td>Optifit, Refill pipet tips, 10 &#956;L</td>
			<td>Sartorius Biohit (Helsinki, Finland)</td>
			<td>790012</td>
			<td></td>
		</tr>
		<tr>
			<td>Optifit, Refill pipet tips, 1,000 &#956;L</td>
			<td>Sartorius Biohit (Helsinki, Finland)</td>
			<td>791002</td>
			<td></td>
		</tr>
		<tr>
			<td>Optifit, Refill pipet tips, 200 &#956;L</td>
			<td>Sartorius Biohit (Helsinki, Finland)</td>
			<td>790202</td>
			<td></td>
		</tr>
		<tr>
			<td>pH glass electrode</td>
			<td>Metrohm (Herisau, Sveits)</td>
			<td>6.0233.100</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter 744</td>
			<td>Metrohm (Herisau, Sveits)</td>
			<td>8.744.1003</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet 10 mL</td>
			<td>Sartorius Biohit (Helsinki, Finland)</td>
			<td>725090</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet m10 &#181;L&#160;</td>
			<td>Sartorius Biohit (Helsinki, Finland)</td>
			<td>725020</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet m100 &#181;L</td>
			<td>Sartorius Biohit (Helsinki, Finland)</td>
			<td>725050</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet m1,000 &#181;L</td>
			<td>Sartorius Biohit (Helsinki, Finland)</td>
			<td>725070</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet m20 &#181;L</td>
			<td>Sartorius Biohit (Helsinki, Finland)</td>
			<td>725030</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl &#8805; 99.9%)</td>
			<td>Sigma Aldrich (St. Louis, MO, USA)</td>
			<td>P-3911</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogenphosphate (pro analysis)</td>
			<td>Merck (Darmstadt, Tyskland)</td>
			<td>1.04873.0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind Eppendorf tube 0.5 mL</td>
			<td>Eppendorf (Hamburg, Tyskland)</td>
			<td>525-0133 (0030 108.094)</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind Eppendorf tube 1.5 mL</td>
			<td>Eppendorf (Hamburg, Tyskland)</td>
			<td>525-0132 (0030 108.116)</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind Eppendorf tube 2.0 mL</td>
			<td>Eppendorf (Hamburg, Tyskland)</td>
			<td>525-0134 (0030 108.450)</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind Eppendorf tube 5.0 mL</td>
			<td>Eppendorf (Hamburg, Tyskland)</td>
			<td>525-0792 (0030108.302)</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Sigma Aldrich (St. Louis, MO, USA)</td>
			<td>Z186716-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide (BioUltra; &#8805; 99.5% )</td>
			<td>Sigma Aldrich (St. Louis, MO, USA)</td>
			<td>71289-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (for analysis)</td>
			<td>Merck (Darmstadt, Tyskland)</td>
			<td>1.06404.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dihydrogenphosphate monohydrate (pro analysis)</td>
			<td>Merck (Darmstadt, Tyskland)</td>
			<td>1.06346.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (AnalaR NORMAPUR)</td>
			<td>VWR International (Radnor, PA, USA)</td>
			<td>28244.295</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium tetraborate decahydrate (&#8805; 99. %)</td>
			<td>Sigma Aldrich (St. Louis, MO, USA)</td>
			<td>S9640-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrafuge Mini Centrifuge</td>
			<td>LABNET International (Edison, NJ, USA)</td>
			<td>C1301</td>
			<td></td>
		</tr>
		<tr>
			<td>Stirring magnet, 25 mm x 6 mm &#216;, circular</td>
			<td>Leybold (Cologne, Germany)</td>
			<td>666&#160;851</td>
			<td></td>
		</tr>
		<tr>
			<td>Stuart&#160;Scientific SA8 vortex mixer</td>
			<td>Stuart (Staffordshire, UK)</td>
			<td>Z648531-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperClear centrifuge tubes (15 mL)</td>
			<td>VWR International (Radnor, PA, USA)</td>
			<td>525-0150</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperClear centrifuge tubes (50 mL)</td>
			<td>VWR International (Radnor, PA, USA)</td>
			<td>525-0155</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermomixer comfort 1.5 mL</td>
			<td>Eppendorf (Hamburg, Tyskland)</td>
			<td>53,55,27,831</td>
			<td>Temperature controlled mixer</td>
		</tr>
		<tr>
			<td>Trizma base (reagent grade, &#8805; 99.0 %)</td>
			<td>Sigma Aldrich (St. Louis, MO, USA)</td>
			<td>T6066</td>
			<td>Tris(hydroxymethyl)aminometan (tris)&#160;</td>
		</tr>
		<tr>
			<td>Trizma HCl (reagent grade, &#8805; 99.0%)</td>
			<td>Sigma Aldrich (St. Louis, MO, USA)</td>
			<td>T3253-100G</td>
			<td>Tris(hydroxymethyl)aminometan HCl (tris HCl)</td>
		</tr>
		<tr>
			<td>Trypsin (TCPK-treated from bovine pancreas, 10,000-15,000 BAEE units/mg Protein)</td>
			<td>Sigma Aldrich (St. Louis, MO, USA)</td>
			<td>T8802</td>
			<td>Store in freezer below -20 &#176;C</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma Aldrich (St. Louis, MO, USA)</td>
			<td>P7949-500ML</td>
			<td>polysorbate 20</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Sigma Aldrich (St. Louis, MO, USA)</td>
			<td>TEM-78511-27</td>
			<td></td>
		</tr>
		<tr>
			<td>Vial caps, white, 9 mm</td>
			<td>Nerliens Meszansky (Oslo, Norge)</td>
			<td>LPP 09 15 0981</td>
			<td></td>
		</tr>
		<tr>
			<td>Mitra microsampler with VAMS (Volumetric adsorptive microsampling) technology, 10 &#181;L, 4-sampler clamshell</td>
			<td>Neoteryx (Torrance, CA, USA)</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

microsampling in targeted mass spectrometry-based protein analysis of low-abundance proteins

This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed using bead-based proteolysis prior to proteotypic peptide affinity-capture and liquid chromatography tandem mass spectrometry (LC-MS/MS) determination. The procedure can be applied to both conventional dried samples using paper cards (e.g., dried blood spots [DBSs] and dried serum spots [DSSs]), as well as samples collected with newer sampling methods such as volumetric absorptive microsampling (VAMS). In addition to describing this procedure, the preparation of both trypsin beads and antibody-coated beads is presented in a step-by-step manner in this work. The advantages of the presented procedure are time-efficient proteolysis using beads and selective robust cleanup using peptide affinity-capture. The current procedure describes the determination of the low-abundance small-cell lung cancer (SCLC) biomarker, progastrin-releasing peptide (ProGRP), in dried serum (both DSSs and VAMS). Detailed procedures for bead preparation make it easier to implement the workflow in new applications or other laboratories. It is demonstrated that the results may be dependent on the sampling material; for the present project, higher signal intensities were seen for samples collected using VAMS compared to DSSs.

An overview of the analytical workflow using both DSS sampling and VAMS is shown in Figure 1. Except for the differences in sampling method, the procedures are identical. Images of the serum sampled using the two sampling methods can be seen in Figure 2.
Both sampling forms (VAMS and DSS) are suitable for the sampling of ProGRP-containing serum. This can be seen from Figure 3 where MS chromatograms of the proteotypic peptide and the IS SIL peptide from DSS and VAMS sampling are shown. In addition, the MS chromatogram after the analysis of a control sample consisting of 10 &#181;L of spiked liquid serum sample processed in the same manner as the dried samples is included. The latter was diluted in the same volume as the volume of the DSS/VAMS extraction solution, subjected to digestion using trypsin beads and cleaned up using peptide affinity capture.
Comparing VAMS and DSS sampling (Figure 4), VAMS provides a higher proteotypic peptide/IS area ratio than DSS. This indicates that there might be a loss of the target protein ProGRP to the paper used for DSS (pure cellulose). When comparing to a control sample, where the serum is not dried prior to further processing and analysis (Figure 4), it is shown that VAMS provides similar area ratios as the control sample (two-tailed t-test, p&#8804; 0.65), indicating no loss to the sampling material, while DSS provides a significantly lower area ratio (two-tailed t-test, p&#8804; 0.005), indicating loss to the sampling material.
A brief evaluation was performed using VAMS. Linearity was shown from 10 to 1,000 ng/mL (R2 = 0.9996), with a limit of detection (LOD, S/N = 3) of 6.7 ng/mL. The LOD is considered satisfactory as the analysis was performed on a rather old triple quadrupole (2008) with a 1 mm ID column. The repeatability of all levels with an S/N &#62; 10 was also considered satisfactory with an RSD between 7% and 17% (n = 3), using IS correction.
Affinity capture can be performed both before and after the digestion step by either capturing the protein of interest or its proteotypic peptide. The current procedure describes peptide affinity capture. An advantage of this approach compared to protein capture is that only the peptide of interest is captured, and an even more efficient sample cleanup is achieved. This is illustrated in Figure 5, showing a more complex full scan chromatogram with more noise after protein capture compared after peptide capture. The samples analyzed in Figure 5 are not sampled using DSS sampling or VAMS; however, serum is also the sample matrix, and affinity capture is performed using the same antibody used for peptide capture in the described procedure.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64473/64473fig01.jpg" /> 
Figure 1: Overview of the analytical workflow using both DSS and VAMS sampling. Abbreviations: DSS = dried serum spot; VAMS = volumetric absorptive microsampling; LC-MS/MS = liquid chromatography-tandem mass spectrometry. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64473/64473fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64473/64473fig02.jpg" /> 
Figure 2: Images of serum sampled using different methods. (A) DBS cellulose card and (B) VAMS. Abbreviations: DBS = dried blood spot; VAMS = volumetric absorptive microsampling. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64473/64473fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64473/64473fig03.jpg" /> 
Figure 3: Representative MS chromatograms of the proteotypic peptide and the IS SIL peptide after DSS and VAMS sampling, as well as for a spiked serum sample added directly into the extraction solution. The MS chromatograms shows 10 &#181;L serum samples spiked with 1.5 &#181;g/mL of ProGRP and applied to (A) VAMS, (B) the cellulose sampling card (for DSS), or (C) directly to the extraction buffer (control sample). Twenty-five microliters of 14 ng/mL IS SIL peptide added to all samples prior to peptide affinity capture. Abbreviations: DSS = dried serum spot; VAMS = volumetric absorptive microsampling; MS = mass spectrometry; ProGRP = progastrin-releasing peptide; IS = internal standard; SIL = stable isotope-labeled. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64473/64473fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64473/64473fig04.jpg" /> 
Figure 4: Representative results of the ALGNQQPSWDSEDSSNFK/IS area ratio for serum samples spiked with ProGRP and applied (10 &#181;L) to VAMS, DSS, or directly to extraction solution (control sample). Concentration of ProGRP is 1.5 &#181;g/mL, n = 4 for each condition; 25 &#181;L of 14 ng/mL IS SIL peptide is added to all samples prior to peptide affinity capture. *indicates that the area ratio is significantly different from samples applied to VAMS (two-tailed t-test, p&#8804; 0.005). Error bars are &#177; standard deviation. Abbreviations: DSS = dried serum spot; VAMS = volumetric absorptive microsampling; ProGRP = progastrin-releasing peptide; IS = internal standard; SIL = stable isotope-labeled. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64473/64473fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64473/64473fig05.jpg" /> 
Figure 5: Comparison of base peak chromatograms (full scan Orbitrap analysis) after intact protein extraction (blue) and proteotypic epitope peptide extraction (red). Extracted ion chromatograms of the proteotypic epitope peptide (ALGNQQPSWDSEDSSNFK, m/z 1005.45) are shown on the right. Serum spiked with 150 ng mL&#8722;1 ProGRP was used as the sample. This figure is reprinted from Levern&#230;s et al.14. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64473/64473fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Table S1: An overview of the composition of buffers and solutions and how to prepare them. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64473/64473_Supp Table S1 Buffers and solutions.xlsx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Microsampling in Targeted Mass Spectrometry-Based Protein Analysis of Low-Abundance Proteins at JoVE.com

Microsampling in Targeted Mass Spectrometry-Based Protein Analysis of Low-Abundance Proteins

A protocol is presented for the determination of low-abundance biomarkers from dried serum samples exemplified with the biomarker progastrin-releasing peptide (ProGRP). Antibody-coated magnetic beads are used for the selective cleanup and enrichment of a proteotypic ProGRP peptide. The captured peptide is subsequently analyzed by liquid chromatography-tandem mass spectrometry.

This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed ...

microsampling-targeted-mass-spectrometry-based-protein-analysis-low

Nanyang Technological University

Research

JoVE Journal

Chemistry

2.3K Views.  University of Oslo. A protocol is presented for the determination of low-abundance biomarkers from dried serum samples exemplified with the biomarker progastrin-releasing peptide (ProGRP). Antibody-coated magnetic beads are used for the selective cleanup and enrichment of a proteotypic ProGRP peptide. The captured peptide is subsequently analyzed by liquid chromatography-tandem mass spectrometry.

Video: Microsampling in Targeted Mass Spectrometry-Based Protein Analysis of Low-Abundance Proteins

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests, novel therapies, and further our understanding of disease processes. The inherent chemical diversity of the metabolome creates significant analytical challenges and there is no single experimental approach that can detect all metabolites. Additionally, the biological variation in individual metabolism and the dependence of metabolism on environmental factors necessitates large sample numbers to achieve the appropriate statistical power required for meaningful biological interpretation. To address these challenges, this tutorial outlines an analytical workflow for large scale non-targeted metabolite profiling of serum by UPLC-MS. The procedure includes guidelines for sample organization and preparation, data acquisition, quality control, and metabolite identification and will enable reliable acquisition of data for large experiments and provide a starting point for laboratories new to non-targeted metabolite profiling by UPLC-MS.

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry UPLC-MS

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to ...

Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass determination allows one to evaluate the correctness of protein primary sequences, the presence of mutations and/or post-translational modifications, the possible protein degradation, the sample homogeneity, and the degree of isotope incorporation in case of labelling (e.g. 13C labelling).
Electrospray ionization (ESI) mass spectrometry (MS) is widely used for mass determination of denatured proteins, but its efficiency is affected by the composition of the sample buffer. In particular, the presence of salts, detergents, and contaminants severely undermines the effectiveness of protein analysis by ESI-MS. Matrix-assisted laser desorption/ionization (MALDI) MS is an attractive alternative, due to its salt tolerance and the simplicity of data acquisition and interpretation. Moreover, the mass determination of large heterogeneous proteins (bigger than 100 kDa) is easier by MALDI-MS due to the absence of overlapping high charge state distributions which are present in ESI spectra.
Here we present an accessible approach for analyzing proteins larger than 100 kDa by MALDI-time of flight (TOF). We illustrate the advantages of using a mixture of two matrices (i.e. 2,5-dihydroxybenzoic acid and &#945;-cyano-4-hydroxycinnamic acid) and the utility of the thin layer method as approach for sample deposition. We also discuss the critical role of the matrix and solvent purity, of the standards used for calibration, of the laser energy, and of the acquisition time. Overall, we provide information necessary to a novice for analyzing intact proteins larger than 100 kDa by MALDI-MS.

Matrix-assisted Laser Desorption/Ionization Time of Flight MALDI-TOF Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Accurate mass measurement represents an important step during the investigation of proteins. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) can be used for such determination and its key advantage is the tolerance to salts, detergents and contaminants. Here we illustrate an accessible approach for the analysis of proteins larger than 100 kDa by MALDI-MS.

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass ...

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique amino acid sequence, it is only realized by the folding of the polypeptide chain into a single defined three-dimensional arrangement or more commonly into an ensemble of interconverting conformations. Investigating the connection between protein conformation and its function is therefore essential for a complete understanding of how proteins are able to fulfill their great variety of tasks. One possibility to study conformational changes a protein undergoes while progressing through its functional cycle is hydrogen-1H/2H-exchange in combination with high-resolution mass spectrometry (HX-MS). HX-MS is a versatile and robust method that adds a new dimension to structural information obtained by e.g. crystallography. It is used to study protein folding and unfolding, binding of small molecule ligands, protein-protein interactions, conformational changes linked to enzyme catalysis, and allostery. In addition, HX-MS is often used when the amount of protein is very limited or crystallization of the protein is not feasible. Here we provide a general protocol for studying protein dynamics with HX-MS and describe as an example how to reveal the interaction interface of two proteins in a complex. &#160;&#160;

Protein conformation and dynamics are key to understanding the relationship between protein structure and function. Hydrogen exchange coupled with high-resolution mass spectrometry is a versatile method to study the conformational dynamics of proteins as well as characterizing protein-ligand and protein-protein interactions, including contact interfaces and allosteric effects.

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique ...

Mass Spectrometric Approaches to Study Protein Structure and Interactions in Lyophilized Powders

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for characterizing lyophilized formulations of protein therapeutics. Labeling followed by suitable proteolytic digestion allows the protein structure and interactions to be mapped with peptide-level resolution. Since the protein structural elements are stabilized by a network of chemical bonds from the main-chains and side-chains of amino acids, specific labeling of atoms in the amino acid residues provides insight into the structure and conformation of the protein. In contrast to routine methods used to study proteins in lyophilized solids (e.g., FTIR), ssHDX-MS and ssPL-MS provide quantitative and site-specific information. The extent of deuterium incorporation and kinetic parameters can be related to rapidly and slowly exchanging amide pools (Nfast, Nslow) and directly reflects the degree of protein folding and structure in lyophilized formulations. Stable photolytic labeling does not undergo back-exchange, an advantage over ssHDX-MS. Here, we provide detailed protocols for both ssHDX-MS and ssPL-MS, using myoglobin (Mb) as a model protein in lyophilized formulations containing either trehalose or sorbitol.

Here, we present detailed protocols for solid-state amide hydrogen/deuterium exchange mass spectrometry (ssHDX-MS) and solid-state photolytic labeling mass spectrometry (ssPL-MS) for proteins in solid powders. The methods provide high-resolution information on protein conformation and interactions in the amorphous solid-state, which may be useful in formulation design.

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 &#181;g of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

Here, we describe a chromatographic assay coupled with the ion mobility separation of peptide precursors followed by the high-resolution (~30,000) MS-detection of peptide fragments for the quantification of spiked peptide standards in a monoclonal antibody digest.

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring ...

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner. In this work, we present a protocol to characterize the exhaled VOCs in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry (Sec-nanoESI-HRMS). The homemade Sec-nanoESI source was readily set up based on a commercial nanoESI source. Hundreds of peaks were observed in the background-subtracted mass spectra of exhaled breath, and the mass accuracy values are -4.0-13.5 ppm and -20.3-1.3 ppm in the positive and negative ion detection modes, respectively. The peaks were assigned with accurate elemental composition according to the accurate mass and isotopic pattern. Less than 30 s is used for one exhalation measurement, and it takes approximately 7 min for six replicated measurements.

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have been thought of as a type of molecular information storage. Mass spectrometry analysis has been considered the method of choice for sequencing peptoids. Peptoids can be synthesized via solid phase chemistry using a repeating two-step reaction cycle. Here we present a method to manually synthesize oligo-peptoids and to analyze the sequence of the peptoids using tandem mass spectrometry (MS/MS) techniques. The sample peptoid is a nonamer consisting of alternating N-(2-methyloxyethyl)glycine (Nme) and N-(2-phenylethyl)glycine (Npe), as well as an N-(2-aminoethyl)glycine (Nae) at the N-terminus. The sequence formula of the peptoid is Ac-Nae-(Npe-Nme)4-NH2, where Ac is the acetyl group. The synthesis takes place in a commercially available solid-phase reaction vessel. The rink amide resin is used as the solid support to yield the peptoid with an amide group at the C-terminus. The resulting peptoid product is subjected to sequence analysis using a triple-quadrupole mass spectrometer coupled to an electrospray ionization source. The MS/MS measurement produces a spectrum of fragment ions resulting from the dissociation of charged peptoid. The fragment ions are sorted out based on the values of their mass-to-charge ratio (m/z). The m/z values of the fragment ions are compared against the nominal masses of theoretically predicted fragment ions, according to the scheme of peptoid fragmentation. The analysis generates a fragmentation pattern of the charged peptoid. The fragmentation pattern is correlated to the monomer sequence of the neutral peptoid. In this regard, MS analysis reads out the sequence information of the peptoids.

A protocol is described for the manual synthesis of oligo-peptoids followed by sequence analysis by mass spectrometry.

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have ...

Analyzing Protein Architectures and Protein-Ligand Complexes by Integrative Structural Mass Spectrometry

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing metabolic reactions, DNA repair and replication. Therefore, a detailed understanding of these processes provides critical information on how cells function. Integrative structural MS methods offer structural and dynamical information on protein complex assembly, complex connectivity, subunit stoichiometry, protein oligomerization and ligand binding. Recent advances in integrative structural MS have allowed for the characterization of challenging biological systems including large DNA binding proteins and membrane proteins. This protocol describes how to integrate diverse MS data such as native MS and ion mobility-mass spectrometry (IM-MS) with molecular dynamics simulations to gain insights into a helicase-nuclease DNA repair protein complex. The resulting approach provides a framework for detailed studies of ligand binding to other protein complexes involved in important biological processes.

Mass spectrometry (MS) has emerged as an important tool for the investigation of structure and dynamics of macromolecular assemblies. Here, we integrate MS-based approaches to interrogate protein complex formation and ligand binding.