The overall goal of this procedure is to develop a technology based on meso porous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. This is accomplished by first fabricating the meso porous silica thin film chips and pretreating with oxygen plasma ashing. The second step is to perform a simple pretreatment of the serum and then to enrich the low molecular weight proteins and peptides from the serum by on-chip fractionation.
Following elucian of the low molecular weight proteins and peptides from the chip, the molecules are profiled using matrix assisted laser desorption ionization time of flight mass spectrometry. The final step is to utilize professional software to perform data analysis. Ultimately, the mass spectra and statistic analysis results reveal the specificity and efficacy of low molecular weight proteome harvesting.
The main advantage of this techniques or exist existing technology like 2D page Western blood, include a powerful capacity to enrich the low abundant low molecular weight proteins and peptides in serum, the high throughput and time efficiency, the reduction of the serum sample requirements and the low cost. One of the most challenging aspect of this procedure is to control a Nanopore net morphology, such as PO size distribution, PO ship, and poor interconnectivity. Because this feature determine the sensitivity and the specificity of a profiling target blood signature.
To ensure exercise, we aim to carefully control the model ratio of starting material and follow the instruction precisely To create the coating solution for the chip. Start by making the hydrolyzed silicate precursor solution. Mix 14 milliliters of teos with 17 milliliters of 200 proof ethanol, 6.5 milliliters of deionized water and 0.5 milliliters of six molar hydrochloric acid under strong stirring.
Continue the strong stirring and heat the resulting solution at 80 degrees Celsius for two hours. Next, prepare the tri block copolymer solution by adding the chosen polymer solution to 10 milliliters of ethanol at room temperature with vigorous stirring. In this demonstration onic F1 27 is used.
Complete the mixture by adding the silicate solution into this tri block copolymer, followed by two hours of strong stirring at room temperature. Apply one milliliter of the resulting coating solution to a four inch silicon wafer by spin coating at a rate of 1, 500 RPM for 20 seconds. Next, heat the wafer at 80 degrees Celsius for 12 hours.
Then raise the temperature one degree Celsius per minute until 425 degrees Celsius is reached and bake at this temperature for five hours. Next, pretreat the resulting meso porous silica or MPS chip surface with oxygen plasma ashing. Then measure the film thickness and porosity using an oxometer.
Meanwhile, to each serum sample, add tri fluoro acidic acid to a final concentration of 0.01%and acetyl nitrile to a final concentration of 5%Then shake these samples on a table vortex shaker at room temperature for 30 minutes subsequent to pre baking of the chips overnight in an oven at 160 degrees Celsius. Use compressed air to dust off any particles that may be on the surface of the chip. Clean the cover slip with 100%ethanol and place on the NPS chip surface, creating the sample wells.
Press the cover slip down with forceps to ensure a complete seal with the chip. Next, pipette 10 microliters of serum sample into each. Well incubate for 30 minutes in a humidified chamber at room temperature to allow the low molecular weight proteins to enter the pores following incubation, aspirate serum from the wells and discard.
Then pipette 10 microliters of deionized water into each well to wash away any larger proteins. Repeat this wash four times working with only one to two wells at a time. To prevent evaporation quickly add five microliters of elution buffer to each sample.
Well pipette up and down 30 times while moving the pipette tip around in the well to mix. After mixing, aspirate all the elucian buffer and place into a micro centrifuge tube until ready to perform matrix assisted laser desorption ionization time of flight analysis to perform maldi toff spot 0.5 microliters of the sample to the MALDI target plate and allow to dry then spot 0.5 microliters of matrix in 50%aceto nitrile containing 0.1%TFA and allow to co crystallize next spot, 0.5 microliters of calibration solution onto calibration spot. Once dry, insert the target plate into the maloff mass spectrometer.
The machine should be sent to positive reflector mode with a laser intensity of 4, 203, 000 shots per sample. First, perform the analysis with a selected mass range of 800 to 5, 000 Daltons with a target mass of 2000 Daltons. Then perform the same MALDI toff analysis in linear mode, but change the mass range to 900 to 10, 000 Daltons or 3000 to 70, 000 Daltons with a target mass of 5, 000 Daltons.
Following multi top analysis process the raw spectra with the convert peak list software, and then export to specal. Align software for pre-processing align all spectra using the peak alignment by fast Fourier transform or P-A-F-F-T correlation method and normalize the intensities to total ion current. Perform the peak detection with a height ratio of two and 0.3%of the mass window.
Correct the baseline and remove the negative values prior to analysis. Importing T 2D files into marker view is the first step for the supervised processing of maldi data of different groups. Start by opening the marker view software, then import the T 2D files.
Select the folder containing the T 2D files and set up the parameters for spectra processing. Next, open the sample table. Highlight samples from the same group, and design a group label.
Normalize the samples using total area sums. Then open the options window. Set up a unique color and symbol for each group.
Then perform principle components analysis and T-test for the active data samples are divided into different groups by PC one and PC two scores. The biomarker candidates would be found according to the PC loading plots show the summary of intensity profile of selected biomarker by clicking plot profiles for selected peaks. The points with the larger values could distinguish different groups.
After comparing groups with a T-test sort the table by ascending P values to determine the peaks that can distinguish various groups shown here are the MS spectra of the serum sample for peptides in the range of 900 to 10, 000 Daltons, and for proteins in the range of 3000 to 70, 000 Daltons. The spectra of the unprocessed samples illustrate the signal suppression in the low molecular weight region due to the presence of well ionized, highly abundant high molecular weight proteins such as albumin after fractionation by the MPS thin films, the majority of the large molecules have been depleted, resulting in a significant enrichment of the low molecular weight components. As a control, the same serum sample was applied onto a non-porous pure silica surface.
To evaluate the specificity of MPS thin films for low molecular weight proteome recovery, there was no significant harvesting of peptides or proteins from the non-porous silica. Thus, it can be concluded that it was the meso porous architecture and not the silica surface affinity that constitutes the predominant factor in the enrichment of LMWP. Precisely controlled variations in pore size can be achieved through the use of copolymers with differing hydrophobic block lengths.
The effect of pore size on the LMW peptide and protein recovery efficacy was investigated using MPS thin films prepared from four onic surfactants with different volume ratios of the hydrophobic and hydrophilic components. Shown here is a magnified view of the maldi spectra demonstrating the characteristic molecular cutoff of each MPS chip correlating to the pore size. The range of pore sizes led to the recovery of a different repertoire of peptides and proteins from the same serum sample via size and shape exclusion.
This can also be observed in the two-way hierarchical clustering of the peptide mix features among the different chips. The intensity of the red or yellow color indicates the relative peptide concentration. Larger pores enhanced the harvesting of larger peptides while the small peptides were preferentially recovered from the chips with smaller pores.
Different MPS thin film periodic nanostructures formed using high molecular weight tri block copolymers, such as onic F1 27 with similar por size distributions can be obtained by tuning the polymer construction in the precursor solution. The 3D cubic and honeycomb hexagonal nanostructures possess more desirable nanopore interconnectivity and more accessible nanopore morphology. As a result, they exhibit superior performance in selectively enriching LMW peptides, then the 2D hexagonal structure.
This is the case even though they share similar pore size distributions and the same molecular cutoff for serum fractionation. The conjugation of Organy lane on MPS chips was streamlined by introducing oxygen plasma ashing to pretreat the chip surface. This figure demonstrates the charge specific recovery for the chips with different surface functions through a bar graph of the MS intensity of detection of selectively captured peptides on the functionalized chips.
According to their isoelectric point, the peptides are positively or negatively charged at pH 7.0 beside the depletion of high molecular weight proteins. The results demonstrate that the structural design and the chemical functionalization of the NPS further increased the specificity of peptide enrichment. With this development.
We expect this technique to pave the way for researcher in the field of proteomics to explore novel biomarkers for early diagnosis or therapeutical evaluation in the body's flute from the human and animal model. Consequently, grid enhanced personalized treatment of major disease.
