Combination of SCX Fractionation and Charge-Reversal Derivatization Facilitates the Identification of Nontryptic Peptides in C-Terminomics

Abstract

The proteome wide, mass spectrometry based identification of protein C-termini is hampered by factors such as poor ionization efficiencies, low yielding labeling strategies, or the need for enrichment procedures. We present a bottom-up proteomics workflow to identify protein C-termini utilizing a combination of strong cation exchange chromatography, on-solid phase charge-reversal derivatization and LC–MS/MS analysis. Charge-reversal improved both MS and MS/MS spectra quality of peptides carrying nonbasic C-terminal residues, allowing the identification of a high number of noncanonical C-termini not identified in nonderivatized samples. Further, we could show that C-terminal 18O labeling introduced during proteolytic processing of the samples is not suitable to distinguish internal from C-terminal peptides. The presented workflow enables the simultaneous identification of proteins by internal peptides and additionally provides data for the C- and N-terminome. Applying the developed workflow for the analysis of a Saccharomyces cerevisiae proteome allowed the identification of 734 protein C-termini in three independent biological replicates, and additional 789 candidate C-termini identified in two or one of three biological replicates, respectively. The developed analytical workflow allowed us to chart the nature of the yeast C-terminome in unprecedented depth and provides an alternative methodology to assess C-terminal proteolytic protein processing.

Mass spectrometry characterization of light chain fragmentation sites in cardiac AL amyloidosis: insights into the timing of proteolysis

“…For the C-terminomics labeling, C-terminal charge-reversal derivatization of the proteins previously subjected to amine protection ( i.e. primary amine derivatization, previously described) was obtained by amidation of the free carboxyl groups with ethanolamine ( 45 , 46 ). For this task, 100 µg of the previously precipitated samples were reconstituted in 100 µl of 0.1 m MES, pH 5/4 m Gnd-HCl (2 h, RT), and carboxyl groups were amidated by adding ethanolamine (final concentration 75 m m , previously buffered in 0.4 m MES, pH 5), EDC-HCl (freshly prepared; final concentration 50 m m ), and NHS (freshly prepared; final concentration 10 m m ).…”

Sequential amidation of peptide C‐termini for improving fragmentation efficiency

“…29 Recently, Kaleja and coworkers reported the derivatization of C-terminal carboxyl groups with N,N-dimethylethylenediamine (DMEDA) to improve MS/MS spectra quality of peptides carrying nontryptic C-terminal residues and the coverage of y-series ions formation under HCD. 30 As previously reported, [31][32][33][34][35] the presence of internal basic residues in peptides could sequester protons and eliminate random protonation as well as bias cleavage sites, thus limiting their effective fragmentation. Collectively, the aforementioned carboxyl derivatization strategies attached positively charged basic groups to peptide C-termini as well as side-chain residues (aspartic, Asp, and glutamic acid, Glu); as a result, internal basic groups in Asp/Glu might have a detrimental effect on the fragmentation behavior of peptide.…”

“…(25, 100, and 1000 mM), the protein coverage (blue bar graph in Figure 2A) was increased from 49.9% to 78.7%, indicating that high concentration MA might promote ionization efficiency, thus benefiting for MS identification. Furthermore, we also tested the effect of different reaction times (10,30,60,90, and 120 min). The significant improvement of methylamidation derivatization efficiency for Cterminal as well as side-chain carboxyl groups could be observed once reaction time reached 120 min, compared with 10-min reaction time (Figure 2B).…”

“…Similarly, Frey et al appended tertiary or quaternary amines to peptide carboxyl groups, generating more highly charged peptide ions that improved ETD peptide fragmentation 29 . Recently, Kaleja and coworkers reported the derivatization of C‐terminal carboxyl groups with N , N ‐dimethylethylenediamine (DMEDA) to improve MS/MS spectra quality of peptides carrying nontryptic C‐terminal residues and the coverage of y‐series ions formation under HCD 30 . As previously reported, 31–35 the presence of internal basic residues in peptides could sequester protons and eliminate random protonation as well as bias cleavage sites, thus limiting their effective fragmentation.…”

One-step Isolation of Protein C-terminal Peptides from V8 Protease-digested Proteins by Metal Oxide-based Ligand Exchange Chromatography

“…14 To date, several methods have been developed to isolate protein C-terminal peptides from digested protein mixtures. [15][16][17][18][19][20][21][22][23][24][25][26][27][28] These methods can be roughly divided into two types, involving positive and negative selection. Positive selection targets protein C-terminal carboxy groups for labeling with oxazolone or carboxypeptidase 16,17 , although the labeling efficiency of these methods is incomplete or biased by the nature of the amino acid residues at the protein C-termini.…”