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. 2010 Jun 15;82(12):5253-9.
doi: 10.1021/ac100651k.

Peptide Orientation Affects Selectivity in Ion-Exchange Chromatography

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Free PMC article

Peptide Orientation Affects Selectivity in Ion-Exchange Chromatography

Andrew J Alpert et al. Anal Chem. .
Free PMC article

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.

Figures

Figure 1
Figure 1
ERLIC of ideal tryptic monophosphopeptide standards; comparison of elution with an increasing salt versus decreasing organic gradient. Column: PolyWAX LP, 100 × 4.6 mm; 5 μm, 300 Å. Flow rate = 1 mL/min. Detection: absorbance; wavelength as noted. Gradient: 0−5′, 0% B; 5−45′, 0−100% B; 45−50′, 100% B. Mobile phase A: 20 mM ammonium formate, pH 2.2, with 70% ACN. Mobile phase B: [top] 100 mM ammonium formate, pH 2.2, with 64% ACN; [bottom] 20 mM ammonium formate, pH 2.2, with 10% ACN. Key: (A) SLYSSSPGGAYVTR (Vimentin(51-64)); (B) SVNFSLTPNEIK (MAP 1B(1271-1281)); (C) WWGSGPSGSGGSGGGK; (A+P) SLYSS(pS)PGGAYVTR; (B+P) SVNFSL(pT)PNEIK; (C+P) WWGSGPSGSGG(pS)GGGK; (D+P) GGAAGLG(pY)LGK. (Inset) Mobile phase A: 20 mM sodium methylphosphonate, pH 2.0, with 70% ACN. Mobile phase B: 200 mM triethylamine phosphate, pH 2.0, with 60% ACN. Gradient: 0−5′, 0% B; 5−25′, 0−100% B. Detection: A220. Column and flow rate: as above. Key: (E) WWGSGP(pS)GSGG(pS)GGGK; (F) WWGSGPSG(pS)GG(pS)GGGK. The double peak for peptide F probably reflects the presence of different conformational isomers around the proline residue.
Figure 2
Figure 2
Orientation of tryptic phosphopeptides in ERLIC with an anion-exchange material (left) and in SCX (right).
Figure 3
Figure 3
Probes of tryptic peptide orientation in ERLIC. Column: PolyWAX LP, 200 × 4.6 mm; 5 μm, 300 Å. Flow rate = 1 mL/min. Detection: 280 nm. Mobile phase (isocratic): 10, 20, or 40 mM ammonium formate, pH 2.2; %ACN as indicated. Key: Top = AAAAAAWNK (red); AAANAAAWK (green); NAAAAAAWK (blue); Bottom = AAAAAAWNK-NH2 (red); AAANAAAWK-NH2 (green); NAAAAAAWK-NH2 (blue); AAAAAAWK-NH2 (purple).
Figure 4
Figure 4
Charge distribution in a regular and an amidated tryptic peptide.
Figure 5
Figure 5
Contribution of a charged residue to retention in SCX as a function of position in a tryptic peptide. Calculations are per ANN comparison of retention times versus sequence (see Materials and Methods for conditions).
Figure 6
Figure 6
Elution in SCX of different classes of peptides in a Lys-N digest. Sample: Lys-N digest of HEK 293T cell lysate. Conditions: See Materials and Methods. (Bottom) Phosphopeptides that eluted in fractions 5−10 had no basic residues or had more than one phosphate group, while N-acetylated peptides eluting later than fraction 18 had extra basic residues. (Top) Distribution of monophosphopeptides with or without extra basic residues in addition to the N-terminal lysine.
Figure 7
Figure 7
Lys-N phosphopeptides: Distribution in SCX fractions as a function of the distance of the phosphate group from the N-terminus. Sample and conditions: See Figure 6. (Top) Peptides with one phosphate group and a Lys residue at the N-terminus but no additional basic residues. The units in the Y-axis are the number of phosphopeptides in the tallest bar in each fraction. (Bottom) Peptides with one phosphate group and a Lys residue at the N-terminus plus at least one additional basic residue. The tallest bar (labeled) corresponds to four peptides. Both graphs share the same X-axis.
Figure 8
Figure 8
Tryptic phosphopeptides: effect of extra phosphate and basic residues on elution in SCX. Sample and conditions (see Figure 6): (Bottom) Peptides with one phosphate group and a Lys/Arg residue at the C-terminus, including peptides with additional basic residues and with segments corresponding to the number of peptides terminating in Arg or Lys residues. (Top) Peptides with additional phosphate groups and basic residues as indicated. The units in the Y-axis are the number of phosphopeptides in the tallest bar in each histogram.
Figure 9
Figure 9
Tryptic phosphopeptides: distribution in SCX fractions as a function of the distance of the phosphate group from the N-terminus (top) or C-terminus (bottom). Sample and conditions: See Figure 6. The graphs include peptides with one phosphate group and a Lys/Arg residue at the C-terminus but no additional basic residues.

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