title: 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.
authors.affiliation: PolyLC Inc., 9151 Rumsey Road, Ste. 180, Columbia, Maryland 21045, USA. aalpert@polylc.com
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Alpert
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Petritis
authors.forename: Lars
authors.initials: L
authors.lastname: Kangas
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
authors.forename: Karl
authors.initials: K
authors.lastname: Mechtler
authors.forename: Goran
authors.initials: G
authors.lastname: Mitulović
authors.forename: Shabaz
authors.initials: S
authors.lastname: Mohammed
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D002852:Chromatography, Ion Exchange / Q000379:methods; D006801:Humans; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
chemical_list: D010455:Peptides; D010710:Phosphates; D014357:Trypsin; D008239:Lysine