Peptide Orientation Affects Selectivity in Ion-Exchange Chromatography

Abstract

Here we demonstrate that separation of proteolytic peptides, having the same net charge and one basic residue, is affected by their specific orientation toward the stationary phase in ion-exchange chromatography. In electrostatic repulsion−hydrophilic interaction chromatography (ERLIC) with an anion-exchange material, the C-terminus of the peptides is, on average, oriented toward the stationary phase. In cation exchange, the average peptide orientation is the opposite. Data with synthetic peptides, serving as orientation probes, indicate that in tryptic/Lys-C peptides the C-terminal carboxyl group appears to be in a zwitterionic bond with the side chain of the C-terminal Lys/Arg residue. In effect, the side chain is then less basic than the N-terminus, accounting for the specific orientation of tryptic and Lys-C peptides. Analyses of larger sets of peptides, generated from lysates by either Lys-N, Lys-C, or trypsin, reveal that specific peptide orientation affects the ability of charged side chains, such as phosphate residues, to influence retention. Phosphorylated residues that are remote in the sequence from the binding site affect retention less than those that are closer. When a peptide contains multiple charged sites, then orientation is observed to be less rigid and retention tends to be governed by the peptide’s net charge rather than its sequence. These general observations could be of value in confirming a peptide’s identification and, in particular, phosphosite assignments in proteomics analyses. More generally, orientation accounts for the ability of chromatography to separate peptides of the same composition but different sequence.

Fluctuations in histone H4 isoforms during cellular reprogramming monitored by middle‐down proteomics

“…2A). This elution profile was in agreement with the one reported in a similar analysis [31] and may reflect the impact of charge position on the peptide orientation in SCX separation mode [43]. Noteworthy, this defined elution profile can also be used to increase the confidence of the localization of the PTM, which is critical in the interpretation of such complex MS/MS spectra (Fig.…”

Enhancing the Identification of Phosphopeptides from Putative Basophilic Kinase Substrates Using Ti (IV) Based IMAC Enrichment

“…2B). The level of results for the direct analysis are largely made possible by the separation of the N-acetylated peptides away from the phosphorylated peptides by SCX (12). Interestingly, the enrichment factor without Ti 4ϩ -IMAC was close to 90% and with enrichment was close to 100%, according to the results generated by Mascot.…”

“…It is estimated that ϳ68% of an in silico tryptic digest of the human IPI protein database generates peptides with a net charge of 1ϩ under pH 2.7 assuming protonation of both the N terminus and the basic residue and deprotonation of the C terminus (9). Such tryptic peptides are often referred to as "2ϩ" in the literature (ourselves guilty) which ignores the deprotonation of the C terminus that has an important impact on the separation (12). Phosphopeptides containing only one basic residue will have a net charge of zero (protonation of the N terminus and the basic residue combined with deprotonation of the C terminus and phosphorylated residue) and will elute earlier in an SCX based separation.…”

“…Typically, low-pH strong cation exchange (SCX) 1 chromatography is used as the first step where peptides are fraction-ated based on their solution net charge (9,10) and the orientation of peptides to the negatively charged chromatographic material (11,12). Unlike glutamic and aspartic acid, phosphorylated amino acids are able to retain a negative charge under acidic (pH 2.7) conditions.…”

Sequential enrichment of singly- and multiply-phosphorylated peptides with zwitterionic hydrophilic interaction chromatography material

“…(SAX) separates phosphopeptides from non-phosphopeptides according to their different solution charge states [12][13][14]. Later, electrostatic repulsion-hydrophilic interaction chromatography (ERLIC), a combination of ion exchange and hydrophilic interaction, was developed for phosphopeptide isolation [15][16][17].…”