On-plate Biological Sample Enrichment And Purification Based On Surface Patter For Mass Spectrometry Analysis

As a simple, rapid, high-throughput and high sensitivity analytical technique,matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has beenwidely used for identification of proteins/peptides in proteomics. However, thedetection of low-abundant and post-translational modified proteins is still a challengeof MALDI MS and a good analytical achievement has been interfered by the use oforganic matrix in the low mass range. Therefore, sample concentration andpurification before MS analysis are typically required. This thesis focused on thedevelopment of a novel method for on-plate biological sample enrichment andpurification in direct MS analysis. Several biological sample pretreatment methodshave been successfully developed based on surface pattern, which should be of greatpotential applications in proteome research.In this thesis, we introduced our works in five chapters.In Chapter1, the proceedings and the challenges of MALDI MS and itsapplications are summarized in brief. Furthermore, we introduced the current researchstatus, the applications and problems and challeges faced on the sample pretreatedmethods in proteome research and surface-assisted laser desorption/ionization massspectrometry (SALDI MS). Based on that, the research intention and purpose of thisthesis was proposed.In Chapter2, a novel technique was proposed to achieve on-plate peptides/proteinsselective enrichment and self-desalting on a hydrophobic-hydrophilic-hydrophobicpatterned sample support for direct MALDI MS analysis. Upon deposition, the sample solution is first concentrated in a small area by repulsion of the hydrophobic outerlayer, and then, the peptides/proteins and coexisting salt contaminants are selectivelycaptured in different regions of the pattern, respectively, resulting in an excellentenrichment and desalting efficiency. As a result, the detection sensitivity is improvedby2orders of magnitude better than the use of the traditional MALDI plate, andhigh-quality mass spectra are obtained even in the presence of NaCl (1M), NH4HCO3(100mM), or urea (1M). The practical application of this method is furtherdemonstrated by the successful analysis of myoglobin digests with high sequencecoverage, demonstrating the great potential in proteomic research.In Chapter3, a novel method has been proposed to achieve selective enrichmentand purification of glycoproteins/glycopeptides on a surface patterned sample support,which consists of a hydrophobic outer layer (F-SAM) and an internal boronicacid-modified gold microspot (900μm). Upon deposition, the sample solution isfirstly concentrated in a small area by repulsion of the hydrophobic outer layer, andthen the glycoproteins/glycopeptides are selectively captured through boronic acidcovalently binding in the inner. However, the non-glycosylated proteins/peptides orhigh concentration salts are removed after rinsing with the alkaline solution. As aresult, the detection sensitivity is improved by an order of magnitude better than theuse of the stainless steel MALDI plate. With the surface patterned sample support, theglycoproteins/glycopeptides can be detected even under the interference from theexcessive existing non-glycosylated proteins/peptides (10times more thanglycoproteins/glycopeptides). Simultaneously, high-quality mass spectra can beobtained even in the presence of urea (1M), NaCl (1M), or NH4HCO3(200mM).Therefore, this novel technique may be applied for high-throughput analysis oflow-abundance glycoproteins/glycopeptides in complicated proteome research.In Chapter4, we have developed the hydrophobic-hydrophilic-hydrophobicpatterned sample support for on-plate peptides enrichment and desalting forsubsequently direct surface-assisted laser desorption/ionization mass spectrometry(SALDI-MS). The outermost hydrophobic layer of the novel sample support is afluorine-terminated SAM, and the inside one is a CHCA-modified SU8(CHCA/SU8)composite. The gap between the SAM and CHCA/SU8is bare silicon that leaves ahydrophilic area. Upon deposition, the salt contaminants and the peptides/proteins notonly can be selectively captured in different areas in one step, but also thepeptides/proteins can be direct detected without the use of organic matrix, which significantly reduced and eliminated the interferences from the matrix ions. As aresult, the detection sensitivity is enhanced20times better than the use of thetraditional MALDI plate, and high-quality mass spectra are obtained even in thepresence of NH4HCO3(100mM), urea (1M) and NaCl (1M). SALDI with on-plateenrichment and desalting approach is successfully applied to peptides analysis fromBSA digests with high sequence coverage, demonstrating the great potential inproteomic research.In conclusion, this thesis aimed at developing several techniques for on-platebiological sample enrichment and purification based on surface pattern for massspectrometry to solve the challenge of low-abundance proteins, post-translationalmodified proteins and matrix interference, which demonstrated their potentialapplication in proteomic research.