Anion and cation mixed-bed ion exchange for enhanced multidimensional separations of peptides and phosphopeptides

Abstract

Shotgun proteomics typically uses multidimensional LC/MS/MS analysis of enzymatically digested proteins, where strong cation-exchange (SCX) and reversed-phase (RP) separations are coupled to increase the separation power and dynamic range of analysis. Here we report an on-line multidimensional LC method using an anion- and cation-exchange mixed bed for the first separation dimension. The mixed-bed ion-exchange resin improved peptide recovery over SCX resins alone and showed better orthogonality to RP separations in two-dimensional separations. The Donnan effect, which was enhanced by the introduction of fixed opposite charges in one column, is proposed as the mechanism responsible for improved peptide recovery by producing higher fluxes of salt cations and lower populations of salt anions proximal to the SCX phase. An increase in orthogonality was achieved by a combination of increased retention for acidic peptides and moderately reduced retention of neutral to basic peptides by the added anion-exchange resin. The combination of these effects led to approximately 100% increase in the number of identified peptides from an analysis of a tryptic digest of a yeast whole cell lysate. The application of the method to phosphopeptide-enriched samples increased by 94% phosphopeptide identifications over SCX alone. The lower pKa of phosphopeptides led to specific enrichment in a single salt step resolving acidic phosphopeptides from other phospho- and non-phosphopeptides. Unlike previous methods that use anion exchange to alter selectivity or enrich phosphopeptides, the proposed format is unique in that it works with typical acidic buffer systems used in electrospray ionization, making it feasible for online multidimensional LC/MS/MS applications.

Figure 1. Proposed mechanism of the ACE mixed-bed system (B) compared to the conventional SCX format (A); a simplified schematic to illustrate the concept based on the Donnan effect for improved peptide recovery from SCX resins and an additional anion-exchange interaction for unique selectivity in the ion-exchange separation

During the peptide binding phase at pH∼2.6 (by the mobile phase), the majority of tryptic peptides are doubly charged and bound to the SCX resin in both systems. When a salt pulse is applied to elute the peptides, salt cations displace peptides by competing with the peptides for SCX's negative charge sites. Salt anions have a mixed effect; the elution of peptides is promoted by neutralization of the peptides' positive charge(s) while the resultant ion-pairs increases peptides' hydrophobicity and discourage elution due to the hydrophobic property of SCX resins. In the ACE mixed-bed format, salt cations and anions are separated inside of the column by the Donnan (exclusion) effect that is enhanced by the fixed opposite charges of both resins. Through repulsion with anion-exchange resins, salt cations are consistently pushed toward the SCX phase resulting in the schematic concentration profile shown in (B) bottom. Conversely, salt anions are pulled away from the SCX surface by the same principle. The net consequence is a greater flux of salt cations and fewer salt anions proximate to the SCX surface compared to the SCX-only format. The displacement of peptides in the ACE mixed-bed format is expected to be facilitated by the sum of those effects. The additional interaction between acidic group(s) of peptides and anion-exchanger resins is unique to the ACE mixed-bed format at the elution phase at pH∼6.8. The ACE mixed-bed format can provide unique selectivity and improved orthogonality by this interaction. The white arrows in the figure refer solely to the direction of the attractive interaction and are not quantitative.

Figure 2. Improved peptide recovery from ion-exchanger resins – chromatographic comparison between SCX and ACE mixed-bed formats

Typical base peak chromatograms and MS/MS spectra from triplicate recovery experiments are shown. Tryptic digests of a five protein standard mixture were eluted from each ion-exchanger column using a 1-step high-concentration salt pulse and subsequently analyzed by online reversed-phase gradient LC/MS/MS. The majority of peptides observed in the ACE mixed-bed platform (B) had greater intensities than those of the SCX format (A). The amounts of packed SCX resin were adjusted to be identical for both cases to make the comparison valid (i.e., the SCX bed was 1/3 length of the WAX+SCX(2:1) bed).

Figure 3. Improved peptide (A) and protein (B) identifications in 13-step MudPIT – the effect of ACE mixed-beds on two-dimensional (2D) LC/MS/MS strategy

0.5-μg of yeast tryptic digests were analyzed by 13-step MudPIT runs for each ion-exchange resin combination (N=4). Peptide/protein identification criteria were: (1) at least 2 peptides identification per protein, (2) peptides must be half- or fully-tryptic, and (3) filtering criteria must result in false positive rates at less than 2% at the protein level as estimated by a decoy reversed-database approach. The ACE mixed-beds are denoted as WAX+SCX(A:B), where WAX and SCX resins were mixed with A:B weight ratios and used as columns for the first-dimension separation. WAX/SCX and SCX/WAX are tandem bed configurations by the packing order of “upstream/downstream” (2.5 cm each in length).

Figure 4. Ion-exchange elution profile comparison of 13-step MudPIT runs with different ion-exchanger combinations

The graphs were generated from the datasets that provided the best peptide/protein identifications in Figure 3 for each resin combination (where applicable). The runs denoted as SCX(half length) (B) and SCX(1/3 length) (C) used columns of half or 1/3 length of the others so that they contained the same amount of SCX resin and could be compared directly to the WAX+SCX(1:1) or (2:1), respectively. Peptide identifications criteria were modified to 1 peptide per protein with estimated false positive rates < 1% at peptide level. The y-axis indicates the number of peptides newly identified in each step so that peptides already identified in previous salt steps were excluded from the profile. Peptides identified in multiple charge states were counted only once.

Figure 5. 2D view plots of 13-step MudPIT runs with different ion-exchanger combinations

The same datasets used in Figure 4 were plotted in two-dimensional planes that consisted of salt step in which the peptide eluted and its RP retention time. Plots (A) and (B) are a comparison of the ACE mixed-bed (WAX+SCX(2:1)) with SCX with an emphasis on the distribution of common and uniquely identified peptides in the two formats. The plots (C)–(F) were generated in the same way except that the identified peptides were categorized according to their theoretical pI.

Figure 6. 2D distribution of phosphopeptides identified in (A) WAX+SCX(2:1) and (B) SCX formats using modified 13-step MudPIT

Phosphopeptides enriched from HeLa cell nuclear extract were analyzed in the both ion-exchanger formats (∼50 μg/run). The phosphopeptides were identified from MS/MS spectra of either the precursor peptide or its neutral loss species, and the plots were generated by combining those identifications. The salt pulse for the Step 13 in this analysis was modified as follows: a 2 min salt pulse of 95% buffer D' + 5% buffer B, where buffer D' was water/acetonitrile (95:5, v/v) containing 500 mM ammonium acetate (pH 1.73 by trifluoroacetic acid). Peptide/protein identification criteria were: (1) one or more peptide identification per protein, (2) peptides must be fully tryptic, and (3) estimated false positive rates less than 0.5% at protein level as determined by a decoy reversed-database approach.