Peptide separation selectivity in proteomics LC‐MS experiments: Comparison of formic and mixed formic/heptafluorobutyric acids ion‐pairing modifiers

• Published in: Journal of separation science Vol. 43; no. 20; pp. 3830 - 3839
• Main Authors: Gussakovsky, Daniel, Anderson, Geoff, Spicer, Vic, Krokhin, Oleg V.
• Format: Journal Article
• Language: English
• Published: Weinheim Wiley Subscription Services, Inc 01.10.2020
• Subjects: Acetonitrile; Amino acids; detection sensitivity; Eluents; Formic acid; heptafluorobutyric acid; Hydrophobicity; Liquid chromatography; Mass spectrometry; Mathematical models; peptide reversed‐phase chromatography; Peptides; Proteomics; Reduction; retention time prediction; Selectivity; Separation
• ISSN: 1615-9306; 1615-9314
• DOI: 10.1002/jssc.202000578

Separation selectivity and detection sensitivity of reversed‐phase high‐performance liquid chromatography with tandem mass spectrometry analyses were compared for formic (0.1%) and formic/heptafluorobutyric (0.1%/0.005%) acid based eluents using a proteomic data set of ∼12 000 paired peptides. The addition of a small amount of hydrophobic heptafluorobutyric acid ion‐pairing modifier increased peptide retention by up to 10% acetonitrile depending on peptide charge, size, and hydrophobicity. Retention increase was greatest for peptides that were short, highly charged, and hydrophilic. There was an ∼3.75‐fold reduction in MS signal observed across the whole population of peptides following the addition of heptafluorobutyric acid. This resulted in ∼36% and ∼21% reduction of detected proteins and unique peptides for the whole cell lysate digests, respectively. We also confirmed that the separation selectivity of the formic/heptafluorobutyric acid system was very similar to the commonly used conditions of 0.1% trifluoroacetic acid, and developed a new version of the Sequence‐Specific Retention calculator model for the formic/heptafluorobutyric acid system showing the same ∼0.98 R2‐value accuracy as the Sequence‐Specific Retention calculator formic acid model. In silico simulation of peptide distribution in separation space showed that the addition of 0.005% heptafluorobutyric acid to the 0.1% formic acid system increased potential proteome coverage by ∼11% of detectable species (tryptic peptides ≥ four amino acids).