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

Abstract

Funding information Natural Sciences and Engineering Research Council of Canada Separation selectivity and detection sensitivity of reversed-phase highperformance 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 ∼3.75fold 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 R 2-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).

Formation of Gaseous Peptide Ions from Electrospray Droplets: Competition between the Ion Evaporation Mechanism and Charged Residue Mechanism

“…74−77 Aqueous droplets are particularly relevant for peptides that are relatively hydrophilic (such as bradykinin) because these species elute early when the acetonitrile content of the LC gradient is low. 7 Our data reveal that both the IEM and the CRM are viable ESI mechanisms for bradykinin. The two pathways are in kinetic competition with one another.…”

“…This is surprising, considering that peptides are the central analytes in bottom-up proteomics. [5][6][7][8][9][10]13 In most LC/ESI-MS workflows, peptides are generated by tryptic digestion. Trypsin cleaves after Arg and Lys (unless followed by Pro), 5,56 generating peptides with ∼10 residues.…”

“…8,57−60 Bradykinin biosynthesis involves hydrolysis of kininogen by plasma kallikrein, a trypsin-like serine protease. 73 Despite the use of water/acetonitrile in LC/ ESI-MS, [7][8][9][10]13 we studied bradykinin in aqueous droplets. This is because organic cosolvents evaporate more quickly than water, such that the final ESI nanodroplets contain a much higher water percentage than the initial solution.…”

Potential of Single Pulse and Multiplexed Drift-Tube Ion Mobility Spectrometry Coupled to Micropillar Array Column for Proteomics Studies

“…Interestingly, the addition of 5% of acetonitrile in the resuspending solvent increased the solubility of most of the hydrophobic peptides and decreased the absorption of these peptides on the surface of polypropylene vials [30]. Since TFA is a well-known ion-pairing agent, used to increase the retention of hydrophilic peptides on an RP stationary phase [31,32], an increased number of identifications could therefore be expected. However, a lower number of identifications was obtained using TFA in the resuspending solvent compared to the presence of FA only.…”

“…However, a lower number of identifications was obtained using TFA in the resuspending solvent compared to the presence of FA only. The retention Since TFA is a well-known ion-pairing agent, used to increase the retention of hydrophilic peptides on an RP stationary phase [31,32], an increased number of identifications could therefore be expected. However, a lower number of identifications was obtained using TFA in the resuspending solvent compared to the presence of FA only.…”