Analysis of a tryptic digest of pig hemoglobin using ESI-FAIMS-MS

The continuous gas-phase ion separation and atmospheric pressure focusing p roperties of high-held asymmetric waveform ion mobility spectrometry (FAIMS ) offer significant advantages for the mass spectrometric analysis of trypt ic digests of proteins. In this study, tryptic peptides of pig hemoglobin w ere examined by ESI-FAIMS-MS using a newly designed FAIMS device. The new, hemispherical geometry of the inner electrode served to deliver the ions, v ia the gas flows, to the center axis of the FAIMS analyzer, improving the s ensitivity relative to previous prototypes. Mass spectra collected using th is new FAIMS showed significantly less chemical background noise than conve ntional ESI-MS, while maintaining approximately the same absolute sensitivi ty as that observed with ESI-MS, As a consequence of the ion separation in FAIMS, the identification of the tryptic fragments was simplified and some peptides, such as the triply protonated VVAGVANALAHK(3+), that were obscure d by the intense background of ESI-MS, were readily detected using ESI-FAIM S-MS. In addition, the FAIMS device was shown to separate isobaric ions at m/z 532.4. Correlations between CV and mass-to-charge ratio, as well as CV and ionic collision cross section, were evaluated for 38 peptide ions ident ified in the tryptic digest, The correlation between the CV of the peptide and the mass-to-charge ratio is very poor, indicating good orthogonality be tween the separation by FAIMS and the separation by mass spectrometry.