Improved recovery of proteome‐informative, protein N‐terminal peptides by combined fractional diagonal chromatography (COFRADIC)

Abstract

We previously described a proteome-wide, peptide-centric procedure for sorting protein N-terminal peptides and used these peptides as readouts for protease degradome and xenoproteome studies. This procedure is part of a repertoire of gel-free techniques known as COmbined FRActional DIagonal Chromatography (COFRADIC) and highly enriches for a-amino-blocked peptides, including a-amino-acetylated protein N-terminal peptides. Here, we introduce two additional steps that significantly increase the fraction of such proteome-informative, N-terminal peptides: strong cation exchange (SCX) segregation of a-amino-blocked and a-amino-free peptides and an enzymatic step liberating pyroglutamyl peptides for 2,4,6-trinitrobenzenesulphonic acid (TNBS) modification and thus COFRADIC sorting. The SCX step reduces the complexity of the analyte mixture by enriching N-terminal peptides and depleting a-amino-free internal peptides as well as proline-starting peptides prior to COFRADIC. The action of pyroglutamyl aminopeptidases prior to the first COFRADIC peptide separation results in greatly diminishing numbers of contaminating pyroglutamyl peptides in peptide maps. We further show that now close to 95% of all COFRADIC-sorted peptides are a-amino-acetylated and, using the same amount of starting material, our novel procedure leads to an increased number of protein identifications.

Stable isotopic labeling in proteomics

“…Third, in the absence of trypsin but at lower pH (e.g., 0.1% TFA as used in conventional RP solvents), slow back-exchange also occurs, which can be avoided by working at higher pH (e.g., using ammonium acetate buffering at pH 4.5-5.5). Fourth, we noticed that for highly acidic peptides, 16 O- 18 O exchange tends to be slow and incomplete. Fifth, the 4 Da spacing requires mass spectrometers with high resolving power (e.g., Q-TOF, Orbitrap, and FT-ICR mass spectrometers) to segregate the isotopic envelopes.…”

“…Our lab introduced the combined fractional diagonal chromatography (COFRADIC) technique to sort amino (N) terminal peptides out of digested proteomes [18,19]. It is essential here to introduce a difference between protein Nterminal peptides and other (internal) peptides so that both types can be distinguished in a diagonal RP-chromatographic setup.…”

“…First, labeling takes place late in the overall analytical process; thus the two samples are processed separately during quite a number of manipulations before being differently labeled and mixed, introducing several sites for technical variation. Second, when trypsin is not fully inactivated or removed after 18 Oexchange, trypsin-mediated back-exchange during downstream processing in solvents containing natural water occurs. Importantly, this isotope dilution is also noticed at acidic pH (from pH 4 to 5 and lower) where trypsin-mediated cleavage of the peptide bond is assumed to be completely halted.…”

“…Fifth, the 4 Da spacing requires mass spectrometers with high resolving power (e.g., Q-TOF, Orbitrap, and FT-ICR mass spectrometers) to segregate the isotopic envelopes. This also implies that the popular ITs can generally not be used for differential proteomics by 18 O-labeling. Although all of this sheds negative light on the technique, trypsinmediated 18 O-tagging has its value, since most issues listed above can be easily controlled and its universal character makes this labeling technique accessible to all types of samples including body fluids.…”

“…Although all of this sheds negative light on the technique, trypsinmediated 18 O-tagging has its value, since most issues listed above can be easily controlled and its universal character makes this labeling technique accessible to all types of samples including body fluids. In addition, intelligent software tools have been developed that are able to tackle variable and inefficient 18 O-labeling (e.g., [32]). …”