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.

Retention Time Prediction for TMT-Labeled Peptides in Proteomic LC-MS Experiments.

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.

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.

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.

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.