A Strategy for Sequencing Peptides from Dilute Mixtures at the Low Femtomole Level Using Membrane Preconcentration-Capillary Electro-Phoresis-Tandem Mass Spectrometry (MPC-CE-MS/MS)

Abstract

A unique method using membrane preconcentration capillary electrophoresis (mPC-CE) on-line with tandem mass spectrometry (mPC-CE-MS/MS) for sequencing <100 fmols of a series of MHC class I synthetic peptides is described. We show how this methodology in conjunction with transient isotachophoresis (tlTP) after analyte elution from the membrane improves conventional CE-MS strategies by permitting the analysis of large sample volumes (>100 μL) while maintaining high analyte resolution and separation efficiencies that are typically afforded by CE. Instrumental parameters, including the internal dimensions of the mPC-CE capillary, collision gas pressure, collision gas type, and collision energy, are all shown to have substantial effect on the abundance of product ions produced in tandem MS/MS spectra when attempting to sequence peptides at the sub 100 femtomole level. Furthermore, we demonstrate that the physico-chemical properties of these analytes can also affect the MS/MS product ion abundance. In particular, the presence of acidic residue tends to reduce mPC-tlTP-CE-MS/MS sensitivity and direct fragmentation processes resulting in incomplete sequence information. This can be overcome by a simple esterification of the carboxylic acid functional group. Ultimately, we present an optimized mPC-tlTP-CE-MS/MS approach that permits peptide sequence determination by consumption of ∼40 femtomole of peptide analyte.