Paradigm Shift: Major Role of Ion-Pairing-Dependent Size Exclusion Effects in Bottom-Up Proteomics Reversed-Phase Peptide Separations.

Can reversed-phase peptide retention be the same for C8 and C18 columns? or increase for otherwise identical columns with a smaller surface area? Can replacing trifluoroacetic acid (TFA) with formic acid (FA) improve the peak shape? According to our common understanding of peptide chromatography, absolutely not. Surprisingly, a thorough comparison of the peptide separation selectivity of 100 and 120 Šfully porous C18 sorbents to maximize the performance of our in-house proteomics LC-MS/MS setup revealed an unexpectedly higher peptide retentivity for a wider pore packing material, despite it having a smaller surface area. Concurrently, the observed increase in peptide retention─which drives variation in separation selectivity between 100 and 120 Špore size materials─was more pronounced for smaller peptides. These findings contradict the central dogmas that underlie the development of all peptide RP-HPLC applications: (i) a larger surface area leads to higher retention and (ii) increasing the pore size should benefit the retention of larger analytes. Based on our intriguing findings, we compared reversed-phase high-performance liquid chromatography peptide retention for a total of 20 columns with pore sizes between 60 and 300 Šusing FA- and TFA-based eluents. Our results unequivocally attest that the larger size of ion pairs in FA- vs TFA-based eluents leads to the observed impact on selectivity and peptide retention. For FA, peptide retention peaks at 200 Špore size, compared to between 120 and 200 Šfor TFA. However, the decrease in retention for narrow-pore particles is more profound in FA. Our findings suggest that common assumptions about analyte size and accessible surface area should be revisited for ion-pair RP separation of small peptides, typical for proteomic applications that are predominantly applying FA eluents. Hybrid silica-based materials with pore sizes of 130-200 Šshould be specifically targeted for bottom-up proteomic applications to obtain both superior peak shape and peptide retentivity. This challenging task of attaining the best RPLC column for proteomics calls for closer collaboration between LC column manufacturers and proteomic LC specialists.

Retention time prediction for post-translationally modified peptides: Ser, Thr, Tyr-phosphorylation.

The development of a peptide retention prediction model for reversed-phase chromatography applications in proteomics is reported for peptides carrying phosphorylated Ser, Thr and Tyr-residues. The major retention features have been assessed using a collection of over 10,000 phosphorylated/non-phosphorylated peptide pairs identified in a series 1D and 2D LC-MS/MS acquisitions using formic acid as ion pairing modifier. Single modification event on average results in increased peptide retention for phosphorylation of Ser (+ 1.46), Thr (+1.33), Tyr (+0.93% acetonitrile, ACN) on gradient elution scale for Luna C18(2) stationary phase. We established several composition and sequence specific features, which drive deviations from these average values. Thus, single phosphorylation of serine results in retention shifts ranging from -2.4 to 5.5% ACN depending on position of the residue, nature of nearest neighbour residues, peptide length, hydrophobicity and pI value, and its propensity to form amphipathic helical structures. We established that the altered ion-pairing environment upon phosphorylation is detrimental for this variability. Hydrophobicity of ion-pairing modifier directly informs the magnitude of expected shifts: (most hydrophilic) 0.5 % acetic acid (larger positive shift upon phosphorylation) > 0.1 % formic acid (positive) > 0.1 % trifluoroacetic (negative) > 0.1 % heptafluorobutyric acid (larger negative shift). The effect of phosphorylation has been also evaluated for several separation conditions used in the first dimension of 2D LC applications: high pH reversed-phase (RP), hydrophilic interaction liquid chromatography (HILIC), strong cation- and strong anion exchange separations.

Compendium of Chromatographic Behavior of Post-translationally and Chemically Modified Peptides in Bottom-Up Proteomic Experiments.

We have systematically evaluated the chromatographic behavior of post-translationally/chemically modified peptides using data spanning over 70 of the most relevant modifications. These retention properties were measured for standard bottom-up proteomic settings (fully porous C18 separation media, 0.1% formic acid as ion-pairing modifier) using collections of modified/nonmodified peptide pairs. These pairs were generated by spontaneous degradation, chemical or enzymatic treatment, analysis of synthetic peptides, or the cotranslational incorporation of noncanonical proline analogues. In addition, these measurements were validated using external data acquired for synthetic peptides and enzymatically induced citrullination. Working in units of hydrophobicity index (HI, % ACN) and evaluating the average retention shifts (ΔHI) represent the simplest approach to describe the effect of modifications from a didactic point of view. Plotting HI values for modified (y-axis) vs nonmodified (x-axis) counterparts generates unique slope and intercept values for each modification defined by the chemistry of the modifying moiety: its hydrophobicity, size, pKa of ionizable groups, and position of the altered residue. These composition-dependent correlations can be used for coarse incorporation of PTMs into models for prediction of peptide retention. More accurate predictions would require the development of specific sequence-dependent algorithms to predict ΔHI values.

Chromatographic behaviour of peptides modified with amine-reacting tags for relative protein quantitation in proteomic applications.

Reversed-phase (RP) HPLC separation of peptides labeled with amine-reacting tags for relative protein quantitation (iTRAQ4, iTRAQ8 - isobaric tag for relative and absolute quantitation, TMT - tandem mass tag) has been investigated using large-scale proteomics derived retention datasets. These tags have similar chemistry but use linkers of different length and hydrophobicity, moving the positively charged functional groups further from peptide backbone. Peptide hydrophobicity (RP HPLC retention), on average, increases in the following order: non-labeled < iTRAQ4 < iTRAQ8 < TMT under both low pH (0.1% formic acid) and pH 10 eluent conditions. At the same time, the interplay between hydrophobicity and length of the labeling group drives the deviations from this order. Thus, longer and less hydrophobic iTRAQ8 moiety results in greater retention increase for peptides carrying amphipathic helical structures at the N-terminus. Development of a peptide retention prediction models for these modifications was achieved by predicting correspondent retention shifts ΔHI (hydrophobicity index,% acetonitrile) between unmodified and labelled peptide pairs.

Peptide retention time prediction for peptides with post-translational modifications: N-terminal (α-amine) and lysine (ε-amine) acetylation.

Development of a peptide retention prediction model in reversed-phase chromatography is reported for acetylated peptides - both N-terminal (α-) and side chain of Lys (ε-amine) residues. Large-scale proteomic 2D LC-MS analyses of acetylated/non-acetylated tryptic digest of whole human cell lysate have been used to assemble representative retention data sets of 25,000+ modified/non-modified pairs. This allowed elucidating chromatographic behaviour of modified peptides in three different separation modes: high pH reversed-phase, HILIC separation on amide phase (first dimension of 2D) and reversed-phase separation with formic acid as ion-pairing modifier in the second dimension. On average, N-terminal acetylation increases peptide RP retention at acidic pH by 5 Hydrophobicity Index units (% acetonitrile). Acetylation of first lysine adds another 4.1%. The magnitude of the retention shift varies greatly depending on the number of modified amines, peptide length, and N-terminal peptide sequence. Large retention shifts have been observed for peptides with hydrophobic N-termini and specifically peptides carrying sequences characteristic for amphipathic helical structures - all in complete agreement with major sequence-specific features of RP retention mechanism. The utility of the modified Sequence Specific Retention Calculator model has been verified for the in-vivo N-terminally acetylated peptides detected by 2D LC-MS/MS analysis of a yeast tryptic digest. The effect of N-terminal acetylation was also evaluated for six different HILIC columns, strong cation- and strong anion exchange separations using previously acquired 2D LC-MS/MS data.

Peptide retention time prediction in hydrophilic interaction liquid chromatography: Zwitter-ionic sulfoalkylbetaine and phosphorylcholine stationary phases.

Peptide retention time prediction models have been developed for zwitter-ionic ZIC-HILIC and ZIC-cHILIC stationary phases (pH 4.5 eluents) using proteomics-derived retention datasets of ~30 thousand tryptic peptides each. Overall, hydrophilicity of these stationary phases was found to be similar to the previously studied Amide HILIC phase, but lower compared to bare silicas. Peptide retention is driven by interactions of all charged (hydrophilic) residues at pH 4.5 (Asp, Glu, Arg, Lys, His), but shows specificity according to orientation of functional groups in zwitter-ionic pair. Thus, ZIC-cHILIC exhibits an increased contribution of negatively charged Asp and Glu due to the distal positioning of positively charged quaternary amines on the stationary phase. These findings confirm that HILIC interactions are driven by both peptide distribution between water layer adsorbed on the stationary phase and by interactions specific to functional groups of the packing material. Sequence-Specific Retention Calculator HILIC models were optimized for these columns showing 0.967-0.976 R2-values between experimental and predicted retention values. ZIC-HILIC separations represent a good choice as a first dimension in 2D LC-MS of peptide mixtures with correlations between retention values of ZIC-HILIC against RPLC found at 0.197 (ZIC-HILIC) and 0.137 (ZIC-cHILIC) R2-values, confirming a good orthogonality.

Separation Orthogonality in Liquid Chromatography-Mass Spectrometry for Proteomic Applications: Comparison of 16 Different Two-Dimensional Combinations.

Peptide separation orthogonality for 16 different 2D LC-ESI MS systems has been evaluated. To compare and contrast the behavior of the first dimension columns, a large proteomic retention data set of ∼30 000 tryptic peptides was collected for each 2D pairing. The selection of the first dimension system was made to cover the most popular peptide separation modes applied in proteomics: reversed-phase (RP) separations with different pH, hydrophilic interaction liquid chromatography (HILIC), strong cation and anion exchange (SCX, SAX), and mixed-mode separations. The separation orthogonality generally increases in the order RP < SCX < HILIC < SAX, with the exception of high pH RP-low pH RP system, which showed the second best orthogonality value (68%), just behind PolySAX LP column (74%). The identification output of the 2D LC-MS/MS system is driven by both separation orthogonality and efficiency, making high pH RP the best choice for the first dimension separation. Its performance in combination with a standard C18 at acidic pH can be increased further through the application of pairwise fraction concatenation. The effect of the latter has been evaluated using in silico fraction concatenation, which has been proven to show improvement only for RP separations in the first dimension. Concatenation of two, three, and four-five fractions into one is shown to be the most effective for high pH RP and HFBA- and TFA-based C18 separations, respectively. We also suggest simple guidelines for the unbiased determination of dissimilarity for two separation dimensions and evaluate separation orthogonality in 3D LC-LC-MS separation space for all systems under investigation.