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Towards an ultimate solution for peptide retention time prediction: the effect of column temperature on separation selectivity

ABSTRACT: The effect of the column temperature on the selectivity of reversed-phase peptide separation in standard separation settings applied in bottom-up proteomics has been evaluated. Tryptic digests of A549, MCF7, Jurkat and HCT116 human cells in non-labeled and TMT-labeled formats have been analyzed by 1D LC-MS/MS at four different column temperature settings: 25, 35, 45 and 55 C. To increase the size of retention datasets available for retention time prediction modelling, 2D LC-MS/MS acquisitions were also performed on a Jurkat digest (both non-labeled and TMT-modified). The first dimension separation featured high pH RPLC separation (XTerra, 1x100 mm column, pH 10, 2 % per minute acetonitrile gradient). Fourteen (non-labeled) and eighteen (TMT) concatenated fractions were analyzed using RPLC-MS settings identical to that of the 1D experiments. The second dimension of RP chromatographic separation employed the Thermo Fisher Scientific EASY Spray column (PepMap, RSLC C18, 2mm, 100-angstrom, 75mm x 50cm), operated at four temperatures with a gradient of 1% to 50% B (A: 100% water, B: 80:20 ACN: water, both containing 0.1% formic acid) in 107 min, followed by a 1 min rapid increase to 100%B and 12 minute wash. The total acquisition time for each sample/fraction was 120 min.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Oleg Krokhin

PROVIDER: MSV000093468 | MassIVE | Wed Nov 22 11:35:00 GMT 2023

REPOSITORIES: MassIVE

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Project description:Recent advances in mass spectrometric (MS) instruments resulting in high mass resolution and high duty cycles have allowed of very deep coverage of proteomes. However, even with state of the art, high duty cycle instruments, the number of proteins identified with a conventional single liquid chromatography-tandem MS (LC/MS/MS) analysis is typically limited and fractionating protein samples prior to LC/MS/MS analysis is crucial for increasing both the analytical dynamic range and proteome coverage. In order to achieve near comprehensive identification of proteomes two-dimensional (2D) chromatography has been an invaluable tool. This first dimension is typically performed with µL/min flow and relatively large column inner diameters, which allow efficient pre-fractionation but typically require peptide amounts in the milligram range while the second dimension is typically performed with nL/min flow rates and capillary columns with small inner diameters. Typically, reverse phase liquid chromatography (RPLC) provides high peak resolution of peptides and achieves higher peak capacities than strong cation exchange chromatography (SCX) due to the faster chromatographic partitioning. Another advantage of RPLC is that the mobile phases used generate much cleaner samples for downstream analyses, while the incorporation of a desalting step for SCX in order to eliminated the high salt concentrations that can significantly decrease the analytical sensitivity of the MS analysis due to ionization problems. When operated at widely different pH values (e.g., 1st dimension pH 10 and 2nd dimension pH 3) 2D RPLC-RPLC provides separation orthogonality comparable to that of the combination of SCX-RPLC. The difference of separation selectivity between the low and high pH (HpH) RPLC comes from the changes in the charge distribution within peptide chains upon altering pH of the eluent. Herein we describe a method in which tryptic peptides are separated in the first dimension by HpH-RPLC and fractions are collected every 90 s over an 80 min gradient. For the second dimension, each of these fractions is individually run by the standard low pH RPLC method. The implementation of the HpH pre-fractionation allows for short second dimension gradients for bottom up LC/MS/MS and yields very deep (e.g. thousands of identifications) protein coverage at reasonable measurement time.

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Towards an ultimate solution for peptide retention time prediction: the effect of column temperature on separation selectivity

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