Chromatographic properties of deamidated peptides with Asn-Gly sequences in proteomic bottom-up experiments.

Studies surrounding deamidation have relied on the chromatographic and mass spectrometric differentiation of Asn containing peptides and their isomeric Asp and isoAsp products. The development of mass spectrometry analytical techniques and characterization of isomer specific fragmentation patterns has permitted the investigation of some deamidation species but has struggled to remain effective when applied and on complex samples or in high throughput scenarios. On the other hand, chromatographic separations can provide additional information to facilitate detection of deamidation.

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.

Retention Time Prediction for Glycopeptides in Reversed-Phase Chromatography for Glycoproteomic Applications.

The sequence-specific retention calculator algorithm (SSRCalc) [ Krokhin , O. V. 2006 , 78 , 7785 ] was adapted for the prediction of retention times of -glycopeptides separated by reversed-phase high performance liquid chromatography (RPLC).

Sequence-Specific Model for Peptide Retention Time Prediction in Strong Cation Exchange Chromatography.

The development of a peptide retention prediction model for strong cation exchange (SCX) separation on a Polysulfoethyl A column is reported. Off-line 2D LC-MS/MS analysis (SCX-RPLC) of S. cerevisiae whole cell lysate was used to generate a retention dataset of ∼30 000 peptides, sufficient for identifying the major sequence-specific features of peptide retention mechanisms in SCX.

3D HPLC-MS with Reversed-Phase Separation Functionality in All Three Dimensions for Large-Scale Bottom-Up Proteomics and Peptide Retention Data Collection.

The growing complexity of proteomics samples and the desire for deeper analysis drive the development of both better MS instrument and advanced multidimensional separation schemes. We applied 1D, 2D, and 3D LC-MS/MS separation protocols (all of reversed-phase C18 functionality) to a tryptic digest of whole Jurkat cell lysate to estimate the depth of proteome coverage and to collect high-quality peptide retention information. We varied pH of the eluent and hydrophobicity of ion-pairing modifier to achieve good separation orthogonality (utilization of MS instrument time).