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 sp ectrometric sensitivity. In order to facilitate such analyses, we have coupled on-line membrane preconcentration-capillary electrophoresis ( mPC-CE) with microspray mass spectrometry (mPC-CE-mu MS) and tandem ma ss spectrometry (mPC-CE-mu MS/MS). Specifically, cell lysate from simi lar to 10(9) EG-7 mouse tumor cells was immunoprecipitated and the rel eased MHC class I peptides were subjected to reverse-phase HPLC. An HP LC fraction containing antigenic peptide(s) shown to induce T-cell sti mulation was subjected to mPC-CE-mu MS. Approximately 10 mu L (from 10 0 mu L) of the fraction was pressure-injected and concentrated on a st yrenedivinylbenzene (SDB) impregnated membrane. The peptides were elut ed from the membrane with similar to 100 nL of 80% methanol, sandwiche d between a leading stcking buffer (LSB, also serving as CE separation medium) of similar to 110 nL of 0.1% acetic acid in 10% methanol, and a trailing stacking buffer (TSB) of similar to 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 sep arated in a Polybrene-coated capillary with application of -20 kV in r everse polarity mode and subsequently sprayed via an emitter coupled t o 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-mu MS/MS and de termined to be SIINFEKL, a peptide (OVA) known to be antigenic in the mouse model system. Sensitivity enhancement over conventional mPC-CE-M S and MS/MS was similar to 100-fold.