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

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-CEμMS/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, sand-wiched 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-CEμMS/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.