Reversed-Phase Liquid Chromatography of Peptides for Bottom-Up Proteomics: A Tutorial

Abstract

The performance of the current bottom-up liquid chromatography hyphenated with mass spectrometry (LC-MS) analyses has undoubtedly been fueled by spectacular progress in mass spectrometry. It is thus not surprising that the MS instrument attracts the most attention during LC-MS method development, whereas optimizing conditions for peptide separation using reversed-phase liquid chromatography (RPLC) remains somewhat in its shadow. Consequently, the wisdom of the fundaments of chromatography is slowly vanishing from some laboratories. However, the full potential of advanced MS instruments cannot be achieved without highly efficient RPLC. This is impossible to attain without understanding fundamental processes in the chromatographic system and the properties of peptides important for their chromatographic behavior. We wrote this tutorial intending to give practitioners an overview of critical aspects of peptide separation using RPLC to facilitate setting the LC parameters so that they can leverage the full capabilities of their MS instruments. After briefly introducing the gradient separation of peptides, we discuss their properties that affect the quality of LC-MS chromatograms the most. Next, we address the in-column and extra-column broadening. The last section is devoted to key parameters of LC-MS methods. We also extracted trends in practice from recent bottom-up proteomics studies and correlated them with the current knowledge on peptide RPLC separation.

Atomistic Details of Peptide Reversed-Phase Liquid Chromatography from Molecular Dynamics Simulations

“…KD scores are widely used for characterizing protein sequences, but they are not reliable predictors of RPLC retention (Figure S2). The reason underlying the limited predictive power of KD scores and related parameters such as logP ,,, is that they only consider the amino acid composition, but not the peptide sequence, structural dynamics, or ion pairing interactions. ,, …”

“…Separation relies on the fact that the analyte migration velocity is modulated by the analyte affinity to the stationary phase. Transient binding to C18 chains will temporarily anchor an analyte to the stationary phase, thereby lowering its migration velocity. ,, The current section examines peptide–C18 interactions that are responsible for differential peptide migration velocities in water/acetonitrile.…”

“…Separation in RPLC is based largely on hydrophobic contacts between the analyte and the stationary phase. , Simply speaking, analytes that bind strongly to the stationary phase elute later, because binding restricts their convective transport in the mobile phase . In reality, the situation is more complex, because RPLC separations employ gradient elution where the mobile phase composition changes over time . The process starts with a high water percentage to promote analyte trapping on the stationary phase close to the column inlet .…”

“…In reality, the situation is more complex, because RPLC separations employ gradient elution where the mobile phase composition changes over time . The process starts with a high water percentage to promote analyte trapping on the stationary phase close to the column inlet . The organic content is then gradually ramped up, such that analytes desorb and migrate through the column.…”

Retention Time Alignment for Protein Turnover Studies Using Heavy Water Metabolic Labeling

“…Metabolic labeling with stable isotopes followed by liquid chromatography coupled with mass spectrometry (LC-MS) is a high-throughput tool to study the turnover of individual proteins in vivo. , LC separates tryptic peptides based on their physicochemical properties (such as hydrophobicity) and reduces the sample complexity. , The positively charged peptides enter the mass spectrometer (MS) which records mass-to-charge ratios ( m / z ) of intact peptides and their isotope profiles in survey scans (MS1). Tandem mass spectra (MS/MS) are acquired in a commonly used data-dependent acquisition (DDA) mode.…”