Chromatographic Isolation of Methionine-containing Peptides for Gel-free Proteome Analysis

Abstract

A novel gel-free proteomic technology was used to identify more than 800 proteins from 50 million Escherichia coli K12 cells in a single analysis. A peptide mixture is first obtained from a total unfractionated cell lysate, and only the methionine-containing peptides are isolated and identified by mass spectrometry and database searching. The sorting procedure is based on the concept of diagonal chromatography but adapted for highly complex mixtures. Statistical analysis predicts that we have identified more than 40% of the expressed proteome, including soluble and membrane-bound proteins. Next to highly abundant proteins, we also detected low copy number components such as the E. coli lactose operon repressor, illustrating the high dynamic range. The method is about 100 times more sensitive than two-dimensional gel-based methods and is fully automated. The strongest point, however, is the flexibility in the peptide sorting chemistry, which may target the technique toward quantitative proteomics of virtually every class of peptides containing modifiable amino acids, such as phosphopeptides, amino-terminal peptides, etc

Novel IEF Peptide Fractionation Method Reveals a Detailed Profile of N-Terminal Acetylation in Chemotherapy-Responsive and -Resistant Ovarian Cancer Cells

“…Long term expression of FKBP5 however, was decreased in the resistant cells compared to the responsive cells. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 COFRADIC 41 or TAILS 32 , the data presented implies that our method is capable to cover Nterminally modified peptides in at least a complementary fashion, as indicated by their entries into the neXtProt database Therefore our method is of particular use when establishing spectral libraries for DIA analysis. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 …”

Stable isotopic labeling in proteomics

“…Only for post-translationally methylated proteins there is a transfer of the 13 C-methyl group from methionine via the activated methyl pathway (see below). Methionyl peptides can be easily isolated using a simple COFRADIC sorting protocol based on oxidation to the more hydrophilic sulfoxide form [23]. Because methionine appears in nearly all eukaryotic proteins at low frequency, targeted isolation of methionyl peptides strongly reduces the overall peptide complexity, while this selection is just focused on those peptides carrying the differential quantitative information.…”

PRIDE: The proteomics identifications database

“…Note that the average increase since January 2004 amounts to 4000 identifications per month, often punctuated by particularly sharp increases when all three machines are fully operational in parallel. The vast majority of the identifications stems from experiments using the COFRADIC gel-free technology [2][3][4].…”