We are grateful to the individuals with BTHS and their families for participating in our research. We thank the Barth Syndrome Foundation US and the Barth Syndrome UK Trust for their support and for helping with the collection of the blood samples at the annual meeting in Bristol. This study was funded by Barth Syndrome Foundation US, Barth Italia Onlus, and Apulia Region.

Blood samples of healthy donors and heart failure patients were collected at the Policlinic Hospital of Bari (Italy), while samples of BTHS patients were obtained by the National Health Service UK BTHS clinic at Bristol Royal Hospital for Children (UK). Written informed consent of healthy donors, patients, and parents (where appropriate) and approvals by the respective ethics committees were obtained.
NOTE: If not used immediately, blood (in K-EDTA gel tube) can be stored at 4 &#176;C for up to 24-48 h.
1. Isolation of leukocytes by dextran sedimentation of red blood cells (RBCs)
<ol>
	<li>Put blood samples (in K-EDTA) on an orbital shaker for 10 min to mix blood.</li>
	<li>To 0.9 mL of whole blood in a 1.5 mL tube, add 0.1 mL of 20% dextran solution (molecular mass &#62; 100 kDa, in 0.9% NaCl). Pipette and disperse the suspension gently 20 times while avoiding air bubbles that would otherwise retain RBCs at the top of the tube. Let RBCs sediment for about 1 h at room temperature.</li>
	<li>Using a syringe, collect and transfer the yellow supernatant to a 15 mL tube, and then centrifuge at 400 x g for 15 min at room temperature (no brake, swing-bucket rotor).</li>
	<li>Discard the supernatant and resuspend the pellet containing mainly leukocytes and residual RBCs in 0.6 mL of ice-cold bi-distilled water (ddH2O).</li>
	<li>After ~15 s, add 0.2 mL of 0.6 M KCl to the cell suspension to restore the correct osmolarity. The short osmotic shock will lyse RBC and leave leukocytes intact. Adjust the final volume to 2.5 mL with 1x PBS.</li>
	<li>Centrifuge the suspension at 400 x g for 15 min at room temperature (no brake, swing-bucket rotor). Discard the supernatant and wash the leukocyte pellet again with 2.5 mL of 1x PBS.</li>
	<li>Centrifuge as in step 1.6 and discard the supernatant. Resuspend the pellet containing leukocytes in 200 &#181;L of sterilized ddH2O. 
	NOTE: In the authors&#39; experience, this corresponds to a protein content ranging from 200 &#181;g to 400 &#181;g.</li>
	<li>Freeze the suspension at -80 &#176;C or directly perform lipid extraction as follows.</li>
</ol>
2. &#34;Mini-extraction&#34; of lipids from isolated leukocytes
<ol>
	<li>Transfer 20 &#181;L of leukocytes suspension (about 20-40 &#181;g proteins) to a 1.5 mL tube and spin at 16,000 x g for 30 s.</li>
	<li>Discard the supernatant, add 10 &#181;L of CHCl3 to the remaining pellet, and pipette repeatedly to promote lipid extraction.</li>
	<li>Finally, add 10 &#181;L of 9-aminoacridine (9-AA) matrix solution (10 mg/mL 9-AA in 2-propanol/acetonitrile, 60:40 v/v) to the pellet in CHCl3. Pipette and disperse repeatedly to mix.</li>
	<li>Spin the solution containing lipids in CHCl3 and 9-AA at 16,000 x g for 30 s, and then deposit the supernatant as droplets of 0.35 &#181;L (three replicates for each sample) on the MALDI target (sample plate) to be analyzed (&#39;dried droplet&#39; deposition method).</li>
	<li>Let the droplets air dry at least for 10-15 min.</li>
</ol>
3. Lipid analysis by MALDI-TOF/MS
<ol>
	<li>Acquire mass spectra of samples in triplicates on a MALDI-TOF mass spectrometer.</li>
	<li>After calibration with lipid standards (see Table of Materials), set the analyses in the negative ion mode and optimize the detection m/z range from 200 Th to 2,000 Th for small molecule analysis.</li>
	<li>Keep the laser fluence 5% above the threshold (of CL and MLCL) to have a S/N (at least 2).</li>
	<li>Acquire spectra in reflector mode using delayed pulsed extraction. For each mass spectrum, average 2,000 single laser shots (sum of 4 x 500). Apply gated matrix suppression to 400 Th to prevent detector saturation.</li>
</ol>
4. How to calculate the (MLCL + CLi)/CLm ratio
<ol>
	<li>Run the MALDI-TOF/MS instrument software (see Table of Materials) to analyze the acquired spectra.</li>
	<li>Using the Open command, open the Spectrum Browser dialog that allows selecting and loading the spectrum of interest.</li>
	<li>On the menu bar, click on the icons Smooth Mass Spectrum and Subtract Mass Spectrum Baseline.</li>
	<li>Click on File &#62; Export &#62; Mass Spectrum and choose the ASCII format for exporting the spectrum as a two-column table with pairs of data points: m/z (x) and intensity (y). Copy and paste the coordinates in a spreadsheet program (*.xls).</li>
	<li>Repeat steps 4.2 to 4.4 for the triplicates of each analyzed sample, pasting the coordinates in the same spreadsheet program file as shown in Figure 1 (Step 1).</li>
	<li>Following the x-ranges shown in Figure 1 (Step 2) for each species listed, calculate the sum of y1, y2, and y3 values (triplicates) by the SUM function to get the peak area (Figure 1; Step 3).</li>
	<li>Perform the average of the triplicates area values by AVERAGE function (Figure 1; Step 3).</li>
	<li>Place the average area values for each species in the column as shown in Figure 1 (Step 2).</li>
	<li>In order to consider only the first isotopologue of the CLm 72:7 species as in28, calculate the isotopic correction for the overlapping between the M + 2 isotopologue of the CLm 72:8 and the monoisotopic peak of CLm 72:7 as shown in Figure 1 (Step 4).</li>
	<li>Finally, calculate the (MLCL + CLi)/CLm ratio as shown in Figure 1 (Step 5).</li>
</ol>

<ol>
	<li>Barth, P. G., 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=X-linked+cardioskeletal+myopathy+and+neutropenia+(Barth+syndrome):+respiratory-chain+abnormalities+in+cultured+fibroblasts.">X-linked cardioskeletal myopathy and neutropenia (Barth syndrome): respiratory-chain abnormalities in cultured fibroblasts.</a> Journal of Inherited Metabolic Disease. 19 (2), 157-160 (1996).</li><li>Steward, C. G., 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=syndrome+(X+linked+cardiac+and+skeletal+myopathy,+neutropenia,+and+organic+aciduria):+rarely+recognised,+frequently+fatal+[abstract].">syndrome (X linked cardiac and skeletal myopathy, neutropenia, and organic aciduria): rarely recognised, frequently fatal [abstract].</a> Archives of Disease in Childhood. 89, 48 (2004).</li><li>Clarke, S. L. N., 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=Barth+syndrome.">Barth syndrome.</a> Orphanet Journal of Rare Diseases. 8, 23 (2013).</li><li>Zegallai, H. M., Hatch, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barth+syndrome:+cardiolipin,+cellular+pathophysiology,+management,+and+novel+therapeutic+targets.">Barth syndrome: cardiolipin, cellular pathophysiology, management, and novel therapeutic targets.</a> Molecular and Cellular Biochemistry. 476 (3), 1605-1629 (2021).</li><li>Taylor, C., 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=Clinical+presentation+and+natural+history+of+Barth+Syndrome:+An+overview.">Clinical presentation and natural history of Barth Syndrome: An overview.</a> Journal of Inherited Metabolic Disease. 45 (1), 7-16 (2022).</li><li>Miller, P. C., Ren, M., Schlame, M., Toth, M. J., Phoon, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bayesian+analysis+to+determine+the+prevalence+of+Barth+syndrome+in+the+pediatric+population.">Bayesian analysis to determine the prevalence of Barth syndrome in the pediatric population.</a> The Journal of Pediatrics. 217, 139-144 (2020).</li><li>Bione, 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=A+novel+X-linked+gene,+G4.5.+is+responsible+for+Barth+syndrome.">A novel X-linked gene, G4.5. is responsible for Barth syndrome.</a> Nature Genetics. 12 (4), 385-389 (1996).</li><li>Whited, K., Baile, M. G., Currier, P., Claypool, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seven+functional+classes+of+Barth+Syndrome+mutation.">Seven functional classes of Barth Syndrome mutation.</a> Human Molecular Genetics. 22 (3), 483-492 (2013).</li><li>Schlame, M., Ren, M., Xu, Y., Greenberg, M. L., Haller, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+symmetry+in+mitochondrial+cardiolipins.">Molecular symmetry in mitochondrial cardiolipins.</a> Chemistry and Physics of Lipids. 138 (1-2), 38-49 (2005).</li><li>Schlame, M., Xu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+function+of+Tafazzin,+a+mitochondrial+phospholipid-lysophospholipid+acyltransferase.">The function of Tafazzin, a mitochondrial phospholipid-lysophospholipid acyltransferase.</a> Journal of Molecular Biology. 432 (18), 5043-5051 (2020).</li><li>Schlame, M., Rua, D., Greenberg, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biosynthesis+and+functional+role+of+cardiolipin.">The biosynthesis and functional role of cardiolipin.</a> Progress in Lipid Research. 39 (3), 257-288 (2000).</li><li>Mileykovskaya, E., Dowhan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiolipin+membrane+domains+in+prokaryotes+and+eukaryotes.">Cardiolipin membrane domains in prokaryotes and eukaryotes.</a> Biochimica et Biophysica Acta. 1788 (10), 2084-2091 (2009).</li><li>Claypool, S. M., Koehler, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complexity+of+cardiolipin+in+health+and+disease.">The complexity of cardiolipin in health and disease.</a> Trends in Biochemical Sciences. 37 (1), 32-41 (2011).</li><li>Ren, M., Phoon, C. K., Schlame, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+and+function+of+mitochondrial+cardiolipin.">Metabolism and function of mitochondrial cardiolipin.</a> Progress in Lipid Research. 55, 1-16 (2014).</li><li>Paradies, G., Paradies, V., Ruggiero, F. M., Petrosillo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+cardiolipin+in+mitochondrial+function+and+dynamics+in+health+and+disease:+Molecular+and+pharmacological+aspects.">Role of cardiolipin in mitochondrial function and dynamics in health and disease: Molecular and pharmacological aspects.</a> Cells. 8 (7), 728 (2019).</li><li>Acoba, M. G., Senoo, N., Claypool, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipid+ebb+and+flow+makes+mitochondria+go.">Phospholipid ebb and flow makes mitochondria go.</a> The Journal of Cell Biology. 219 (8), 03131 (2020).</li><li>Schlame, M., 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=Phospholipid+abnormalities+in+children+with+Barth+syndrome.">Phospholipid abnormalities in children with Barth syndrome.</a> Journal of the American College of Cardiology. 42 (11), 1994-1999 (2003).</li><li>Wortmann, S. B., 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=Inborn+errors+of+metabolism+with+3-methylglutaconic+aciduria+as+discriminative+feature:+proper+classification+and+nomenclature.">Inborn errors of metabolism with 3-methylglutaconic aciduria as discriminative feature: proper classification and nomenclature.</a> Journal of Inherited Metabolic Disease. 36 (6), 923-928 (2013).</li><li>Ikon, N., Ryan, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+origin+of+3-methylglutaconic+acid+in+disorders+of+mitochondrial+energy+metabolism.">On the origin of 3-methylglutaconic acid in disorders of mitochondrial energy metabolism.</a> Journal of Inherited Metabolic Disease. 39 (5), 749-756 (2016).</li><li>Kulik, W., 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=Bloodspot+assay+using+HPLC-tandem+mass+spectrometry+for+detection+of+Barth+syndrome.">Bloodspot assay using HPLC-tandem mass spectrometry for detection of Barth syndrome.</a> Clinical Chemistry. 54 (2), 371-378 (2008).</li><li>Vaz, F. M., 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=An+improved+functional+assay+in+blood+spot+to+diagnose+Barth+syndrome+using+the+monolysocardiolipin/cardiolipin+ratio.">An improved functional assay in blood spot to diagnose Barth syndrome using the monolysocardiolipin/cardiolipin ratio.</a> Journal of Inherited Metabolic Disease. 45 (1), 29-37 (2022).</li><li>Bowron, A., 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=Diagnosis+of+Barth+syndrome+using+a+novel+LC-MS/MS+method+for+leukocyte+cardiolipin+analysis.">Diagnosis of Barth syndrome using a novel LC-MS/MS method for leukocyte cardiolipin analysis.</a> Journal of Inherited Metabolic Disease. 36 (5), 741-746 (2013).</li><li>Sun, G., 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=Matrix+assisted+laser+desorption/ionization+time-of-flight+mass+spectrometric+analysis+of+cellular+glycerophospholipids+enabled+by+multiplexed+solvent+dependent+analyte-matrix+interactions.">Matrix assisted laser desorption/ionization time-of-flight mass spectrometric analysis of cellular glycerophospholipids enabled by multiplexed solvent dependent analyte-matrix interactions.</a> Analytical Chemistry. 80 (19), 7576-7585 (2008).</li><li>Leopold, J., Popkova, Y., Engel, K. M., Schiller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+of+useful+MALDI+matrices+for+the+mass+spectrometric+characterization+of+lipids.">Recent developments of useful MALDI matrices for the mass spectrometric characterization of lipids.</a> Biomolecules. 8 (4), 173 (2018).</li><li>Angelini, R., Babudri, F., Lobasso, S., Corcelli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI-TOF/MS+analysis+of+archaebacterial+lipids+in+lyophilized+membranes+dry-mixed+with+9-aminoacridine.">MALDI-TOF/MS analysis of archaebacterial lipids in lyophilized membranes dry-mixed with 9-aminoacridine.</a> The Journal of Lipid Research. 51 (9), 2818-2825 (2010).</li><li>Angelini, R., 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=Lipidomics+of+intact+mitochondria+by+MALDI-TOF+MS.">Lipidomics of intact mitochondria by MALDI-TOF MS.</a> The Journal of Lipid Research. 53 (7), 1417-1425 (2012).</li><li>Angelini, R., Vormieter, G., Corcelli, A., Fuchs, 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+fast+method+for+the+determination+of+PC/LPC+ratio+in+intact+horse+serum+by+MALDI-TOF-MS:+an+easy-to-follow+lipid+biomarker+of+inflammation.">A fast method for the determination of PC/LPC ratio in intact horse serum by MALDI-TOF-MS: an easy-to-follow lipid biomarker of inflammation.</a> Chemistry and Physics of Lipids. 183, 169-175 (2014).</li><li>Angelini, R., 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=Cardiolipin+fingerprinting+of+leukocytes+by+MALDI-TOF/MS+as+a+screening+tool+for+Barth+syndrome.">Cardiolipin fingerprinting of leukocytes by MALDI-TOF/MS as a screening tool for Barth syndrome.</a> The Journal of Lipid Research. 56 (9), 1787-1794 (2015).</li><li>Lobasso, 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=A+lipidomic+approach+to+identify+potential+biomarkers+in+exosomes+from+melanoma+cells+with+different+metastatic+potential.">A lipidomic approach to identify potential biomarkers in exosomes from melanoma cells with different metastatic potential.</a> Frontiers in Physiology. 12, 748895 (2021).</li><li>Angelini, R., 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=Visualizing+cholesterol+in+the+brain+by+on-tissue+derivatization+and+quantitative+mass+spectrometry+imaging.">Visualizing cholesterol in the brain by on-tissue derivatization and quantitative mass spectrometry imaging.</a> Analytical Chemistry. 93 (11), 4932-4949 (2021).</li><li>Greco, V., 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=Applications+of+MALDI-TOF+mass+spectrometry+in+clinical+proteomics.">Applications of MALDI-TOF mass spectrometry in clinical proteomics.</a> Expert Review of Proteomics. 15 (8), 683-696 (2018).</li><li>Duncan, M., DeMarco, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI-MS:+Emerging+roles+in+pathology+and+laboratory+medicine.">MALDI-MS: Emerging roles in pathology and laboratory medicine.</a> Clinical Mass Spectrometry (Del Mar, Calif). 13, 1-4 (2019).</li></ol>

All authors declare that the study was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.

Barth syndrome is an inborn error of metabolism and a life-changing condition that is likely to be under-diagnosed2,6. As mentioned before, a contributing factor may be the lack of a straightforward diagnostic test. Here, a simple and fast method to measure MLCL/CL ratio by MALDI-TOF/MS in leukocytes for BTHS screening was described. Moreover, MALDI-TOF mass spectrometers are widely distributed among clinical laboratories worldwide and do not require high analytical expertise31,32.
The current method has a major advantage over existing approaches in that it can simultaneously detect in a single run of MS analysis the CL and MLCL species in the total lipid profile of isolated leukocytes, avoiding time-consuming steps of standard lipid extraction and separation via HPLC. In addition to the time factor, having more steps in the workflow can potentially introduce errors. The present method is easy even for operators without relevant experience in lipidomics.
The method for BTHS screening described here not only relies on the decrease of CLm levels but also considers the increase of CLi species, which along with the presence of MLCL species specifically suggest the CL metabolism defect, i.e., mutations in the TAFAZZIN gene. This approach is successful owing to the use of 9-AA as the matrix for MALDI analysis, which is particularly suitable for the detection of the CL species23,25, even when these species are minor components in complex lipid profiles.
Comparing these results to those obtained in LC/MS studies on leukocytes or bloodspots20,21,22, the mean MLCL/CL ratio in BTHS patients is around 10 in all reports. On the contrary, the mean ratio in controls varies and it is found to be ~0.001 in22, 0.01 in21, and finally 0.1 in28. The reason for the last value being slightly higher is the conservative approach undertaken in the calculations28. Indeed, the method previously reported in28 and the one described here include measurements for MLCL and CLi also in controls&#39; mass spectra, where these are nearly absent. This in fact means accounting for noise levels, which artificially increase the ratio in healthy controls. The reason for this cautionary approach is that low-performance MALDI-MS instruments may not be sensitive enough to detect low levels of the lipids of interest. By considering noise levels, this method assumes a basal level of undetectable MLCL and CLi in controls, and of CLm in patients. Notably, even with such a precautional approach, the application of the MLCL/CL ratio yields a &#62;10-fold difference in the MLCL/CL ratio between BTHS patients and control groups (see Figure&#160;4). Therefore, the assay described here maintains 100% sensitivity and specificity21,22,28.
One limitation of this method is in that it employs 1 mL of blood, although it can be scaled down to 500 &#181;L in practice. This is certainly a drawback when compared to LC/MS methods employing bloodspots, where the latter can be shipped with regular mail and are usually made of ~50 &#181;L of blood. However, this MALDI method has the major advantage of being performable by operators without previous experience in MS analyses, and within only 2 h from withdrawal.
In summary, the proposed assay is simple and can be easily integrated into the routine work of a clinical biochemistry laboratory. This could potentially increase the number of samples analyzable, hence contributing to the identification of new hidden cases.

Barth syndrome (BTHS) is a rare X-linked disease characterized by early-onset cardiomyopathy, skeletal muscle myopathy, growth delay, neutropenia, variable mitochondrial respiratory chain dysfunction, and abnormal mitochondrial structure1,2,3,4,5. BTHS has a prevalence of one case per million males with currently less than 250 known cases worldwide, though it is widely accepted that the disease is underdiagnosed2,6. BTHS results from loss-of-function mutations of the Tafazzin (TAFAZZIN) gene localized to chromosome Xq28.127,8 causing deficient remodeling of the mitochondrial phospholipid cardiolipin (CL), a process that normally leads to a highly symmetric and unsaturated acyl composition9,10. CL has been considered the signature lipid of mitochondria, where it is an important constituent of the inner membrane, vital for oxidative phosphorylation (i.e., mitochondrial energy metabolism), supercomplex formation, protein import, and involved in mitochondrial dynamics, mitophagy, and apoptosis11,12,13,14,15,16. Upon TAFAZZIN loss-of-function, CL remodeling fails and specific phospholipid abnormalities arise in mitochondria of BTHS patients: mature CL level (CLm) is decreased, while increased levels of monolysocardiolipin (MLCL) and altered CL acyl composition (i.e., immature CL species, CLi) occur. This brings to a dramatic increase of the MLCL/CL ratio17.
Diagnosis of BTHS is often difficult, as the disorder presents extremely variable clinical and biochemical features and may differ between affected individuals from the same family and within a patient over time3,5. Many BTHS boys show a very high level of urinary excretion of 3-methylglutaconic acid (3-MGCA), but the urine level may be normal or only mildly increased in patients over time3. However, increased 3-MGCA is a feature of various other mitochondrial and non-mitochondrial disorders, such as 3-methylglutaconyl-CoA hydratase deficiency (AUH defect), 3-methylglutaconic aciduria, dystonia-deafness, encephalopathy, Leigh-like (MEGDEL) syndrome, Costeff syndrome, and dilated cardiomyopathy with ataxia (DCMA) syndrome18,19. Hence, the poor specificity of 3-MCGA as a marker for BTHS and the enormous variability in patients render the biochemical diagnosis ambiguous.
Moreover, over 120 different TAFAZZIN mutations have been described causing the disorder5 and, therefore, a genetic diagnosis can be complicated, slow, and expensive. Moreover, molecular analysis of the TAFAZZIN gene can lead to false-negative results in the presence of mutations in noncoding or regulating sequences3. BTHS can be unambiguously tested by determining the relative amounts and distribution of (monolyso-)CL species and confirmed by TAFAZZIN gene sequencing or vice versa.
A practical test for diagnosis is the measurement of the MLCL/CL ratio by High-Performance Liquid Chromatography (HPLC) and Electro Spray Ionization / Mass spectrometry (ESI/MS) analysis in blood spot20,21. Measuring CL level alone is not adequate for diagnosis as some patients have near-normal levels of CL but altered MLCL/CL ratio. Therefore, measurement of MLCL/CL ratio has 100% sensitivity and specificity for BTHS diagnosis21. Another validated method based on HPLC and ESI/MS analysis has been set up on leukocytes22, but the complex chromatographic techniques for separation of lipids previously extracted and the expensiveness of the instruments restrict this analysis to a few clinical laboratories. All these factors, together with the lack of a straightforward diagnostic test, have contributed to the under-diagnosis of the condition.
MALDI-TOF/MS is a further valid tool in lipid analysis23,24.This analytical technique can be used to directly obtain lipid profiles of various biological samples, thus skipping extraction and separation steps25,26,27,28,29, including in tissue sections for MS Imaging applications30. Given this advantage, a simple and fast method to diagnose BTHS by profiling mitochondrial CL in intact leukocytes with MALDI-TOF/MS was developed28. Leukocyte isolation from only 1 mL of whole blood by erythrocyte sedimentation and lysis is straightforward and does not require special equipment or reagents. Furthermore, a fast lipid &#34;mini-extraction&#34; protocol applicable to minute amounts of leukocytes was described to warrant the successful acquisition of spectra having cleaner MS signals with a higher signal-to-noise ratio (S/N) than in those obtained from intact leukocytes28. This further step takes little time and allows for analyses to be reproducible even when carried out on MS instruments with poor sensitivity. In summary, the analytical method described here requires minimal sample preparation because time-consuming and labor-intensive chromatographic lipid separation can be skipped, thereby speeding up the test.

<table><tbody><tr><td>1,1&amp;prime;,2,2&amp;prime;-tetratetradecanoyl cardiolipin</td><td>Avanti Polar Lipids</td><td>750332</td><td>Lipid standard for MALDI-TOF calibration</td></tr><tr><td>1,1&amp;prime;2,2&amp;prime;-tetra- (9Z-octadecenoyl) cardiolipin</td><td>Avanti Polar Lipids</td><td>710335</td><td>Lipid standard for MALDI-TOF calibration</td></tr><tr><td>1,2-di- (9Z-hexadecenoyl)-sn-glycero-3-phosphoethanolamine</td><td>Avanti Polar Lipids</td><td>878130</td><td>Lipid standard for MALDI-TOF calibration</td></tr><tr><td>1,2-ditetradecanoyl-sn-glycero-3-phosphate</td><td>Avanti Polar Lipids</td><td>830845</td><td>Lipid standard for MALDI-TOF calibration</td></tr><tr><td>1,2-ditetradecanoyl-snglycero-3-phospho-(1&amp;prime;-rac-glycerol)</td><td>Avanti Polar Lipids</td><td>840445</td><td>Lipid standard for MALDI-TOF calibration</td></tr><tr><td>1,2-ditetradecanoyl-sn-glycero-3-phospho-L-serine</td><td>Avanti Polar Lipids</td><td>840033</td><td>Lipid standard for MALDI-TOF calibration</td></tr><tr><td>2-Propanol, ACS reagent, &amp;ge;99.5%</td><td>Merck Life Science S.r.l.</td><td>190764</td><td></td></tr><tr><td>9-Aminoacridine hemihydrate, 98%</td><td>Acros Organics</td><td>134410010</td><td></td></tr><tr><td>Acetonitrile, ACS reagent, &amp;ge;99.5%</td><td>Merck Life Science S.r.l.</td><td>360457</td><td></td></tr><tr><td>Chloroform, ACS reagent, &amp;ge;99.8%</td><td>Merck Life Science S.r.l.</td><td>319988</td><td></td></tr><tr><td>Dextran from Leuconostoc spp. Mr 450,000-650,000</td><td>Merck Life Science S.r.l.</td><td>31392</td><td></td></tr><tr><td>Flex Analysis 3.3</td><td>Bruker Daltonics</td><td></td><td>Software</td></tr><tr><td>MALDI-TOF mass spectrometer Microflex LRF</td><td>Bruker Daltonics</td><td></td><td></td></tr><tr><td>Microsoft Excel</td><td>Microsoft Office</td><td></td><td>Software</td></tr><tr><td>OmniPur 10X PBS Liquid Concentrate</td><td>Merck Life Science S.r.l.</td><td>6505-OP</td><td></td></tr><tr><td>Potassium chloride, ACS reagent, 99.0-100.5%</td><td>Merck Life Science S.r.l.</td><td>P3911</td><td></td></tr><tr><td>Sodium chloride, ACS reagent, &amp;ge;99.0%</td><td>Merck Life Science S.r.l.</td><td>S9888</td><td></td></tr></tbody></table>

fingerprinting cardiolipin in leukocytes by mass spectrometry for a rapid diagnosis of barth syndrome

Cardiolipin (CL), a dimeric phospholipid carrying four fatty acid chains in its structure, is the lipid marker of mitochondria, wherein it plays a crucial role in the functioning of the inner membrane. Its metabolite monolysocardiolipin (MLCL) is physiologically nearly absent in the lipid extract of animal cells and its appearance is the hallmark of the Barth syndrome (BTHS), a rare and often misdiagnosed genetic disease that causes severe cardiomyopathy in infancy. The method described here generates a "cardiolipin fingerprint" and allows a simple assay of the relative levels of CL and MLCL species in cellular lipid profiles. In the case of leukocytes, only 1 mL of blood is required to measure the MLCL/CL ratio via matrix-assisted laser desorption ionization - time-of-flight/mass spectrometry (MALDI-TOF/MS) just within 2 h from blood withdrawal. The assay is straightforward and can be easily integrated into the routine work of a clinical biochemistry laboratory to screen for BTHS. The test shows 100% sensitivity and specificity for BTHS, making it a suitable diagnostic test.

In this study, a simple and rapid method for isolating leukocytes from 1 mL of whole blood and obtaining CL fingerprinting by MALDI-TOF/MS has been described (see Figure 2). Figure 3 shows the comparison of representative CL fingerprinting of leukocytes, obtained from control subjects and BTHS young boys, in the CL and MLCL mass (m/z) range. Table 1 lists CL and MLCL species detected in these mass spectra.
Defects in the TAFAZZIN gene typically determine a lipid profile specific for the BTHS, i.e., the appearance of MLCL and CLi forms together with the reduction of CLm forms in the CL fingerprint. The lipid profile of leukocytes of control subjects typically exhibits only two molecular species of CLm (upper panel of Figure 3). The only peaks of interest present in this mass spectrum are CLm species: tetralinoleoyl CL (18:2)4 at m/z 1,448.0 and CL (18:2)3 (18:1)1 at m/z 1,450.0. This method allows their visualization in the MALDI lipid profile so that the two CL species can be easily identified and measured in the healthy phenotype.
The representative CL fingerprinting of a boy affected by BTHS shows a different pattern of MALDI signals (lower panel of Figure 3). The signals attributable to CLm species are lower, while peaks assigned to two CLi species (at m/z 1,404.0 and 1,430.0) and two MLCL species (at m/z 1,165.8 and 1,191.8) are also found (see Table 1). In summary, data in Figure 3 clearly show that the mitochondrial CL and MLCL species can be readily detected by MALDI-TOF/MS analyses of leukocytes.
A previous study has demonstrated that the (MLCL + CLi)/CLm ratio calculated in lipid profiles of leukocytes obtained by MALDI-TOF/MS can be used as a diagnostic parameter for BTHS28. In that study, CL fingerprinting of leukocytes isolated from healthy subjects and BTHS boys was compared28. Here, results obtained from the MALDI lipid profile of leukocytes isolated from 11 pediatric heart failure patients (one BTHS-affected and 10 non-BTHS-affected) were added.
Figure 4 shows calculated (MLCL + CLi)/CLm ratios in MALDI mass spectra of lipid mini-extracts of leukocytes from nine BTHS patients, 10 non-BTHS-affected (i.e., heart failure patients), and 22 healthy subjects; the ratio has been calculated by considering the area of CL and MLCL peaks detected in the mass spectra, as described, but intensities can also be used with similar results (data not shown).
The (MLCL + CLi)/CLm ratio for BTHS-patients was 7.2 &#177; 2.9, while those calculated for healthy subjects and heart failure patients were 0.19 &#177; 0.10 and 0.13 &#177; 0.07, respectively (mean &#177; SD). It can be seen that control groups (both healthy subjects and heart failure patients) and BTHS-patients are separated by more than one order of magnitude, showing that this method has strong diagnostic power.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63552/63552fig01.jpg" /> 
Figure 1: (MLCL+CLi)/CLm ratio&#160;determination. The calculation of the (MLCL + CLi)/CLm ratio is performed using the Excel program. Step 1: Table of m/z (x) / intensity (y) for species of interest (in triplicates). Step 2: Table of x-ranges to calculate each peak area. Step 3: Calculation of SUM and AVERAGE of y1, y2, and y3 values (triplicates). Step 4: Calculation of isotopic correction. Step 5: Calculation of the (MLCL+CLi)/CLm ratio. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63552/63552fig01large.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/63552/63552fig02.jpg" /> 
Figure 2: Work-flow chart. From the blood withdrawal to the test results, only 2 h are necessary. MALDI-TOF/MS-based BTHS diagnosis in few steps: (1) Blood withdrawal; (2) Dextran sedimentation of erythrocytes, (3) residual erythrocytes lysis (pp1) and leukocytes collection (pp2) by centrifugation, (4) lipid &#34;mini-extraction&#34;, (5) sample deposition, (6) MALDI-TOF/MS analysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63552/63552fig02large.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/63552/63552fig03.jpg" /> 
Figure 3: BTHS diagnostic leukocyte CL fingerprints. Comparison of cardiolipin fingerprints of leukocytes isolated from a control subject (CTRL) and a representative Barth syndrome patient (BTHS) is shown. Red bands highlight peaks of the immature form of cardiolipin (CLi) and monolysocardiolipin (MLCL) species, while the blue band marks the peaks of the mature forms of CL (CLm). Signals labeled with an asterisk refer to gangliosides and are not of interest to this study. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63552/63552fig03large.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/63552/63552fig04.jpg" /> 
Figure 4: (MLCL + CLi)/CLm ratios in controls and BTHS patients. Error bars indicate standard deviations obtained from the mean of measurements of all subjects for each of the three groups: nine BTHS patients, 10 non-BTHS-affected heart failure patients, and 22 healthy subjects. Each sample was analyzed by MALDI-TOF/MS (in triplicate). The y-axis shows a logarithmic scale. The upper cut-off value of controls, calculated as in references22,28 is 0.4. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63552/63552fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>m/z [M-H]-</td>
			<td>Assignment</td>
		</tr>
		<tr>
			<td>1165.8</td>
			<td>MLCL 52:2</td>
		</tr>
		<tr>
			<td>1191.8</td>
			<td>MLCL 54:1</td>
		</tr>
		<tr>
			<td>1404.0</td>
			<td>CLi 68:2</td>
		</tr>
		<tr>
			<td>1430.0</td>
			<td>CLi 70:3</td>
		</tr>
		<tr>
			<td>1448.0</td>
			<td>CLm 72:8</td>
		</tr>
		<tr>
			<td>1450.0</td>
			<td>CLm 72:7</td>
		</tr>
	</tbody>
</table>
Table 1: List of detected peaks assigned to CL and MLCL species in the MALDI-TOF mass spectra.

Watch this Scientific Journal Video about Fingerprinting Cardiolipin in Leukocytes by Mass Spectrometry for a Rapid Diagnosis of Barth Syndrome at JoVE.com

Fingerprinting Cardiolipin in Leukocytes by Mass Spectrometry for a Rapid Diagnosis of Barth Syndrome

This protocol shows how to obtain a mass spectrometric "fingerprint" of leukocyte cardiolipin for the diagnosis of Barth syndrome. The assessment of elevated monolysocardiolipin to cardiolipin ratio discriminates patients with Barth syndrome from control heart failure patients with 100% sensitivity and specificity.

Cardiolipin (CL), a dimeric phospholipid carrying four fatty acid chains in its structure, is the lipid marker of mitochondria, wherein it plays a crucial ...

fingerprinting-cardiolipin-leukocytes-mass-spectrometry-for-rapid

Nanyang Technological University

Research

JoVE Journal

Biochemistry

2.3K Views.  Swansea University. This protocol shows how to obtain a mass spectrometric "fingerprint" of leukocyte cardiolipin for the diagnosis of Barth syndrome. The assessment of elevated monolysocardiolipin to cardiolipin ratio discriminates patients with Barth syndrome from control heart failure patients with 100% sensitivity and specificity.

Video: Fingerprinting Cardiolipin in Leukocytes by Mass Spectrometry for a Rapid Diagnosis of Barth Syndrome

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis&#8211;mass spectrometry (CE-MS). CE is well-suited for the analysis of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. A recently developed sheathless interfacing design, i.e., a porous tip interface, is used for coupling CE to electrospray ionization (ESI) MS. This interfacing approach allows the effective use of the intrinsically low-flow property of CE in combination with MS, resulting in nanomolar detection limits for a broad range of polar metabolite classes. The protocol presented here is based on employing a bare fused-silica capillary with a porous tip emitter at low-pH separation conditions for the analysis of a broad array of metabolite classes in biological samples. It is demonstrated that the same sheathless CE-MS method can be used for the profiling of cationic metabolites, including amino acids, nucleosides and small peptides, or anionic metabolites, including sugar phosphates, nucleotides and organic acids, by only switching the MS detection and separation voltage polarity. Highly information-rich metabolic profiles in various biological samples, such as urine, cerebrospinal fluid and extracts of the glioblastoma cell line, can be obtained by this protocol in less than 1 hr of CE-MS analysis.

A protocol for metabolic profiling of biological samples by capillary electrophoresis&#8211;mass spectrometry using a sheathless porous tip interface design is presented.

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

Profiling the Triacylglyceride Contents in Bat Integumentary Lipids by Preparative Thin Layer Chromatography and MALDI-TOF Mass Spectrometry

The mammalian integument includes sebaceous glands that secrete an oily material onto the skin surface. Sebum production is part of the innate immune system that is protective against pathogenic microbes. Abnormal sebum production and chemical composition are also a clinical symptom of specific skin diseases. Sebum contains a complex mixture of lipids, including triacylglycerides, which is species-specific. The broad chemical properties exhibited by diverse lipid classes hinder the specific determination of sebum composition. Analytical techniques for lipids typically require chemical derivatizations that are labor-intensive and increase sample preparation costs. This paper describes how to extract lipids from mammalian integument, separate broad lipid classes by thin-layer chromatography, and profile the triacylglyceride contents using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This robust method enables a direct determination of the triacylglyceride profiles among species and individuals, and it can be readily applied to any taxonomic group of mammals.

Mammalian integument contains solvent-extractable lipids that can provide chemical compositions characteristic of individual species. This paper presents a routine method for separating broad lipid classes isolated from integumentary tissues using thin layer chromatography and determining the triacylglyceride profile by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The mammalian integument includes sebaceous glands that secrete an oily material onto the skin surface. Sebum production is part of the innate immune ...

Chemistry

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological tissues at the hundreds to tens of microns scale. When performed under ambient conditions, sample pre-treatment becomes unnecessary, thus simplifying the protocol while maintaining the high quality of information obtained. Desorption electrospray ionization (DESI) is a spray-based ambient MSI technique that allows for the direct sampling of surfaces in the open air, even in vivo. When used with a software-controlled sample stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species' spatial distribution. The fidelity of the DESI-MSI output depends on the source orientation and positioning with respect to the sample surface and mass spectrometer inlet. Herein, we review how to prepare tissue sections for DESI imaging and additional experimental conditions that directly affect image quality. Specifically, we describe the protocol for the imaging of rat brain tissue sections by DESI-MSI.

Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient method by which samples, including biological tissues, can be imaged with minimal sample preparation. By rastering the sample below the ionization probe, this spray-based technique provides sufficient spatial resolution to discern molecular features of interest within tissue sections.

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological ...

Bioengineering

Dried Blood Spot Collection of Health Biomarkers to Maximize Participation in Population Studies

Biomarkers are directly-measured biological indicators of disease, health, exposures, or other biological information. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spots meet this need, and can be collected in the field with high response rates. These elements are particularly important in longitudinal study designs including interventions where attrition is critical to avoid, and high response rates improve the interpretation of results. Dried Blood Spot sample collection is simple, quick, relatively painless, less invasive then venipuncture, and requires minimal field storage requirements (i.e.&#160;samples do not need to be immediately frozen and can be stored for a long period of time in a stable freezer environment before assay). The samples can be analyzed for a variety of different analytes, including cholesterol, C-reactive protein, glycosylated hemoglobin, numerous cytokines, and other analytes, as well as provide genetic material. DBS collection is depicted as employed in several recent studies.

Biomarkers are directly-measured biological indicators of disease or health. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spot (DBS) collection meets this need, can be collected in the field with high response rates&#160;and analyzed for a variety of biomarkers.

Biomarkers are directly-measured biological indicators of disease, health, exposures, or other biological information. In population and social sciences, ...

Medicine

Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will die from the disease. The human leukocyte antigen (HLA) class I samples peptides derived from proteasomal degradation of cellular proteins and presents these fragments on the cell surface for interrogation by circulating cytotoxic T lymphocytes (CTL). Generation of T-cell receptor mimic (TCRm) monoclonal antibodies (mAbs) which recognize breast cancer specific peptide/HLA-A*02:01 complexes such as those derived from macrophage migration inhibitory factor (MIF19-27) and NY-ESO-1157-165 enable detection and destruction of breast cancer cells in the absence of an effective anti-tumor CTL response. Intact class I HLA/peptide complexes are shed by breast cancer cells and represent potentially relevant cancer biomarkers. In this work, a breakthrough biomarker screening system for cancer diagnostics incorporating T-cell receptor mimic monoclonal antibodies combined with a novel, label-free biosensor utilizing guided-mode resonance (GMR) sensor technology is presented. Detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated. The impact of this work could revolutionize personalized medicine through development of companion disease diagnostics for targeted immunotherapies.

Intact class I HLA/peptide complexes are shed by cancer cells, representing a potential relevant cancer biomarker. Utilizing label-free sensor technology and T-cell receptor mimicking monoclonal antibodies, detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated, enabling development of a novel cancer diagnostic platform.

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will ...

Immunology and Infection

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Biology

Use of Capillary Electrophoresis Immunoassay to Search for Potential Biomarkers of Amyotrophic Lateral Sclerosis in Human Platelets

Capillary electrophoresis immunoassay (CEI), also known as capillary western technology, is becoming a method of choice for screening disease relevant proteins and drugs in clinical trials. Reproducibility, sensitivity, small sample volume requirement, multiplexing antibodies for multiple protein labeling in the same sample, automated high-throughput ability to analyze up to 24 individual samples, and short time requirement make CEI advantageous over the classical western blot immunoassay. There are some limitations of this method, such as the inability to utilize a gradient gel (4%&#8211;20%) matrix, high background with unrefined biological samples, and commercial unavailability of individual reagents. This paper describes an efficient method for running CEI in a multiple assay setting, optimizing protein concentration and primary antibody titration in one assay plate, and providing user-friendly templates for sample preparation. Also described are methods for measuring pan TDP-43 and phosphorylated TDP-43 derivative in platelet lysate cytosol as part of the initiative in blood-based biomarker development for neurodegenerative diseases.

Blood-based biomarkers for neurodegenerative diseases are essential for implementing large-scale clinical studies. A reliable and validated blood test should require a small sample volume as well as be a less invasive sampling method, affordable, and reproducible. This paper demonstrates that high-throughput capillary electrophoresis immunoassay satisfies criteria for potential biomarker development.

Capillary electrophoresis immunoassay (CEI), also known as capillary western technology, is becoming a method of choice for screening disease relevant ...

Neuroscience

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.	
Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Measuring biomarkers in complex biological samples is increasingly guiding clinical decision-making. We describe a highly sensitive method to simultaneously measure cardiac myosin binding protein-C, creatine kinase MB, and cardiac troponin I in serum samples from subjects with myocardial infarction and healthy control subjects.

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...