Enrichment by organomercurial agarose and identification of cys-containing peptides from yeast cell lysates.
Dynamic range and the presence of highly abundant proteins limit the number of proteins that may be identified within a complex mixture. Cysteine (Cys) has unique chemical reactivity that may be exploited for chemical tagging/capture with biotin/avidin reagents or affinity chromatography allowing specific isolation and subsequent identification of peptide sequences by mass spectrometry. Organomercurial agarose (Hg-beads) specifically captures Cys-containing peptides and proteins from cell lysates. Tryptic peptides from yeast lysates containing Cys were captured and eluted from Hg-beads after incubation with TCEP and trypsin. From two 1 h nano 1-D LC DDA/MS of the eluate >700 proteins were identified with an estimated false positive rate of approximately 1%. Few peptides were identified with high confidence without Cys within their sequence after capture, and extensive washing, indicating little nonspecific binding. The number of fragmentation spectra was increased using automated 2-D nano-LC/MS and allowed identification of 1496 proteins with an estimated false positive rate of 1.1%. Approximately 4% of the proteins identified were from peptides that did not contain Cys, and these were biased toward higher abundance proteins. Comparison of the 1496 proteins to those reported previously showed that >25% were from yeast proteins not previously observed. Most proteins were identified from a single peptide, and sequence coverage was sacrificed by focusing only on identifying Cys-containing peptides, but large numbers of proteins were rapidly identified by eliminating many of the peptides from the higher abundance proteins.
Raftery
Mark J
MJ
Bioanalytical Mass Spectrometry Facility, University of New South Wales, Sydney, New South Wales 2052, Australia.
D002846:Chromatography, Affinity / Q000379:methods; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D009941:Organomercury Compounds / Q000737:chemistry*; D010720:Phosphines / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D012685:Sepharose / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
D003545:Cysteine; D009941:Organomercury Compounds; D010720:Phosphines; D029701:Saccharomyces cerevisiae Proteins; D012685:Sepharose; D014357:Trypsin; C080938:tris(2-carboxyethyl)phosphine