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 ∼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.