Enhanced sensitivity for sequence determination of major histocompatibility complex class I peptides by membrane preconcentration ‐ capillary electrophoresis ‐microspray ‐ tandem mass spectrometry

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Abstract
Sequence analysis of antigenic major histocompatibility complex (MHC) class I peptides requires minimizing sample loss and enhancing mass spectrometric sensitivity. In order to facilitate such analyses, we have coupled on‐line membrane preconcentration‐capillary electrophoresis (mPC‐CE) with microspray mass spectrometry (mPC‐CE‐μMS) and tandem mass spectrometry (mPC‐CE‐μMS/MS). Specifically, cell lysate from ∼ 109 EG‐7 mouse tumor cells was immunoprecipitated and the released MHC class I peptides were subjected to reverse‐phase HPLC. An HPLC fraction containing antigenic peptide(s) shown to induce T‐cell stimulation was subjected to mPC‐CE‐μMS. Approximately 10 μL (from 100 μL) of the fraction was pressure‐injected and concentrated on a styrenedivinylbenzene (SDB) impregnated membrane. The peptides were eluted from the membrane with ∼100 nL of 80% methanol, sandwiched between a leading stcking buffer (LSB, also serving as CE separation medium) of ∼110 nL of 0.1% acetic acid in 10% methanol, and a trailing stacking buffer (TSB) of ∼ 110 nL of 0.1% NH4OH. On application of the CE voltage the peptides are subjected to moving boundary transient isotachophoresis and focused. The peptides were separated in a Polybrene‐coated capillary with application of −20 kV in reverse polarity mode and subsequently sprayed via an emitter coupled to the CE capillary by a liquid junction containing a platinum wire. An ion at m/z 482.3 was detected and subjected to mPC‐CE‐μMS/MS and determined to be SIINFEKL, a peptide (OVA) known to be antigenic in the mouse model system. Sensitivity enhancement over conventional mPC‐CE‐MS and MS/MS was ∼100‐fold.

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