RESUMO
We studied the effect of the column temperature on the selectivity of reversed-phase peptide separation in bottom-up proteomics. The number of peptide identifications from 2 h liquid chromatography with tandem mass spectrometry (LC-MS/MS) acquisitions reaches a plateau at 45-55 °C, driven simultaneously by improved separation efficiency, a gradual decrease in peptide retention, and possible on-column degradation of peptides at elevated temperatures. Performing 2D LC-MS/MS acquisitions at 25, 35, 45, and 55 °C resulted in the identification of â¼100,000 and â¼120,000 unique peptides for nonmodified and tandem mass tags (TMT)-labeled samples, respectively. These peptide collections were used to investigate the temperature-driven retention features. The latter is governed by the specific temperature response of individual residues, peptide hydrophobicity and length, and amphipathic helicity. On average, peptide retention decreased by 0.56 and 0.5% acetonitrile for each 10 °C increase for label-free and TMT-labeled peptides, respectively. This generally linear response of retention shifts allowed the extrapolation of predictive models beyond the studied temperature range. Thus, (trap) column cooling from room temperature to 0 °C will allow the retention of an additional 3% of detectable tryptic peptides. Meanwhile, the application of 90 °C would result in the loss of â¼20% of tryptic peptides that were amenable to MS/MS-based identification.
Assuntos
Peptídeos , Espectrometria de Massas em Tandem , Cromatografia Líquida/métodos , Temperatura , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida de Alta Pressão/métodos , Peptídeos/químicaRESUMO
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.
Assuntos
Peptídeos , Proteômica , Proteômica/métodos , Cromatografia Líquida de Alta Pressão/métodos , Peptídeos/química , Cromatografia de Fase Reversa/métodosRESUMO
We present the first detailed study of chromatographic behavior of peptides labeled with tandem mass tags (TMT and TMTpro) in 2D LC for proteomic applications. Carefully designed experimental procedures have permitted generating data sets of over 100,000 nonlabeled and TMT-labeled peptide pairs for the low pH RP in the second separation dimension and data sets of over 10,000 peptide pairs for high-pH RP, HILIC (amide and silica), and SCX separations in the first separation dimension. The average increase in peptide RPLC (0.1% formic acid) retention upon TMT labeling was found to be 3.3% acetonitrile (linear water/acetonitrile gradients), spanning a range of -4 to 10.3%. In addition to the bulk peptide properties such as length, hydrophobicity, and the number of labeled residues, we found several sequence-dependent features mostly associated with differences in N-terminal chemistry. The behavior of TMTpro-labeled peptides was found to be very similar except for a slightly higher hydrophobicity: an average retention shift of 3.7% acetonitrile. The respective versions of the sequence-specific retention calculator (SSRCalc) model have been developed to accommodate both TMT chemistries, showing identical prediction accuracy (R2 â¼ 0.98) for labeled and nonlabeled peptides. Higher retention for TMT-labeled peptides was observed for high-pH RP and HILIC separations, while SCX selectivity remained virtually unchanged.
Assuntos
Proteômica , Espectrometria de Massas em Tandem , Acetonitrilas/química , Cromatografia Líquida , Peptídeos/análise , Proteômica/métodosRESUMO
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.
Assuntos
Lisina , Proteômica , Acetilação , Aminas , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Humanos , Peptídeos , Processamento de Proteína Pós-Traducional , Espectrometria de Massas em TandemRESUMO
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 â¼30â¯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.
Assuntos
Peptídeos/análise , Proteômica/métodos , Animais , Bovinos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa , Interações Hidrofóbicas e Hidrofílicas , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Espectrometria de Massas em Tandem , Tripsina/metabolismoRESUMO
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.