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-microMS) and tandem mass spectrometry (mPC-CE-microMS/MS). Specifically, cell lysate from approximately 10(9) 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-microMS. Approximately 10 microL (from 100 microL) of the fraction was pressure-injected and concentrated on a styrenedivinylbenzene (SDB) impregnated membrane. The peptides were eluted from the membrane with approximately 100 nL of 80% methanol, sandwiched between a leading stacking buffer (LSB, also serving as CE separation medium) of approximately 110 nL of 0.1% acetic acid in 10% methanol, and a trailing stacking buffer (TSB) of approximately 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-microMS/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 approximately 100-fold.
Biomedical Mass Spectrometry Facility and Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905, USA. naylor.stephen@mayo.edu
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S
Naylor
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Ji
K L
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Johnson
A J
AJ
Tomlinson
W C
WC
Kieper
S C
SC
Jameson
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D019075:Electrophoresis, Capillary / Q000379:methods*; D006583:Hexadimethrine Bromide; D015395:Histocompatibility Antigens Class I / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D009374:Neoplasms, Experimental / Q000276:immunology; D012680:Sensitivity and Specificity; D017421:Sequence Analysis; D013601:T-Lymphocytes / Q000276:immunology; D014407:Tumor Cells, Cultured
D015395:Histocompatibility Antigens Class I; D006583:Hexadimethrine Bromide