Posttranslational protein phosphorylation helps control many cellular processes, so identification of stimuli-induced phosphorylation events is vital to understanding cell regulation. Characterization of phosphorylated proteins by mass spectrometry (MS) typically employs small phosphopeptides (<3000 Da) generated by complete protein digestion. However, efficient phosphopeptide ionization for MS and tandem MS (MS/MS) analysis requires enrichment of phosphopeptides from complex protein digests. Alternatively, digestion of proteins to larger peptides (middle-down proteolysis) can improve protein sequence coverage in MS analysis and reveal a wider range of phosphorylation sites than complete digestion. Initially, this dissertation reviews recent studies on phosphopeptide enrichment and middle-down proteolysis. Based on these studies, the research aims to (1) improve enrichment of small phosphopeptides for MS analysis of target phosphoproteins, and (2) generate large tryptic peptides for middle-down proteomics.
In studies on enrichment, polymer brushes derivatized with nitrilotriacetate-Fe(III) complexes (NTA-Fe) or oxotitanium (oxoTi) capture phosphopeptides from protein digests. Specifically, polymer microspots derivatized with NTA-Fe and surrounded by hydrophobic poly(dimethylsiloxane) facilitate pre-concentration and enrichment of dilute phosphopeptides from only 1 µL of digest. Matrix-assisted laser desorption/ionization (MALDI)-MS directly on the 250-µm spots leads to a sub-fmol detection limit for a β-casein phosphopeptide. Compared to films derivatized with NTA-Fe, polymer-oxoTi brushes improve the capture and detection of mono-phosphopeptides in mixtures of model protein digests. Moreover, MALDI collision-induced dissociation (CID)-MS/MS on polymer-oxoTi identified unknown phosphopeptides from several p65-associated phosphoproteins. These phosphoproteins immunoprecipitated together with p65 from the nuclear extracts of human acute monocytic leukemia cells.
Bifunctionalization of polymer brushes with both oxoTi and NTA-Fe groups leads to simultaneous low-bias enrichment of mono- and multi-phosphorylated peptides from moderately complex model protein digests. For larger samples (tens of µLs), stacking two membranes modified with polymer-oxoTi and polymer-NTA brushes, respectively, in an HPLC fitting enhances phosphopeptide enrichment. The two membranes sequentially capture mono- and multi-phosphorylated peptides from 3 phosphoprotein digests (∼1 µg total mass) in the presence of ∼70 µg of non-phosphoprotein digest.
To control digestion for middle-down MS analysis of proteins, pumping protein solutions through nylon membranes containing immobilized trypsin affords digestion times ranging from a few milliseconds to seconds. In modified membranes with 0.45 µm pores, a 50-ms digestion of apomyoglobin leads to peptides with MWs primarily between 2.5 and 6 kDa. Five of these peptides cover the entire protein sequence. A 10-ms β-casein residence time in modified membranes with 1.2-µm pores yields peptides with masses primarily between 3 and 20 kDa. Two large peptides constitute the entire protein sequence, and one of them covers all the five phosphorylation sites. These simple enrichment methods and new digestion techniques for middle-down proteomics should facilitate future studies of protein phosphorylation.
|Advisor:||Bruening, Merlin L.|
|Commitee:||Blanchard, Gary J., Reid, Gavin E., Tepe, Jetze J.|
|School:||Michigan State University|
|School Location:||United States -- Michigan|
|Source:||DAI-B 72/07, Dissertation Abstracts International|
|Subjects:||Analytical chemistry, Biochemistry|
|Keywords:||Mass spectrometry, Phosphopeptide enrichment, Polymer brushes, Protein phosphorylation, Proteolysis|
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