Electrophoresis zyxwvutsrqponmlkjihg 1998, zyxwvutsrqponm 19, 2201-2212 Sequence determination of MHC zyxw class I peptides 2207 Stephen Naylor', zyxwvutsrqp * Qinchung Ji' Kenneth L. Johnson' Andy J. Tomlinson' William C. Kiepe8 Stephen C. Jameson3 'Biomedical Mass Spectrometry Facility and Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA *Department of Pharmacology and Clinical Pharmacology Unit, Mayo Clinic, Rochester, MN, USA 3Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN, USA 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 mem- brane preconcentration-capillary electrophoresis (mPC-CE) with microspray mass spectrometry (mPC-CE-pMS) and tandem mass spectrometry (mPC-CE- pMS/MS). Specifically, cell lysate from - lo9 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-pMS. Approxi- mately 10 pL (from 100 pL) of the fraction was pressure-injected and concen- trated on a styrenedivinylbenzene (SDB) impregnated membrane. The pep- tides 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% NH,OH. On application of the CE voltage the peptides are subjected to moving boundary transient isotachopho- resis and focused. The peptides were separated in a Polybrene-coated capillary with application of -20 kV in reverse polarity mode and subsequently sprayed zy via an emitter coupled to the CE capillary by a liquid junction containing a pla- tinum wire. An ion at zyxwvu mlz 482.3 was detected and subjected to mPC-CE- pMS/MS and determined to be SIINFEKL, a peptide (OVA) known to be antigenic in the mouse model system. Sensitivity enhancement over conven- tional mPC-CE-MS and MS/MS was - 100-fold. 1 Introduction In order to overcome the poor concentration limits of detection (CLOD) associated with capillary electro- phoresis (CE) we have previously described and devel- oped membrane preconcentration-CE (mPC-CE) [ 1-31. A Teflon cartidge containing a membrane impregnated with a suitable chromatographic phase is installed at the inlet end of the capillary as shown in Fig. 1. This method has also been demonstrated to be compatible with on-line mass spectrometry (mPC-CE-MS) [4-121. By using this approach, sample contaminants such as biolo- gical matrix components or chemical reagents can be pre-eluted from the impregnated membrane phase. This on-line cleanup reduces sample handling, improves ana- lyte recovery and allows the direct analysis of druglxeno- biotic metabolites, peptides, proteins, and other biopo- lymers derived from biological tissue and fluids zyxwvu [Z, 10, 111. Correspondence: Dr. S. Naylor, BMSF, Guggenheim CL C009B, Mayo Clinic, Rochester, MN 55905, USA (Tel: +507-284-5220; Fax: +507-284- 8433; E-mail: naylor.stephen@mayo.edu) Abbreviations: CLOD, concentratio limits of detection; LSB, leading stacking buffer; MHC, major histocompatibility complex; mPC-CE- MS, membrane preconcentration - capillary electrophoresis - mass spectrometry; mPC-CE-pMS, membrane preconcentration - capillary electrophoresis - microspray mass spectrometry; MSIMS, tandem mass spectrometry; SDB, styrenedivinylbenzene, tlTP, transient iso- tachophoresis; TSB, trailing stacking buffer Keywords: On-line preconcentration / Capillary electrophoresis / Transient isotachophoresis / Polybrene / Microspray mass spectro- metry / Peptide sequencing More recently we have described the use of mPC-CE- MS, and tandem MS (mPC-CE-MUMS), to charac- terize and sequence complex mixtures of major histo- compatibility complex (MHC) class I peptides [8, 10, 13-15]. These peptides are important signals of the immune system. Certain exogenous (nonself) MHC class I peptides that are presented at the cell surface can elicit a cytolytic-T-lymphocyte (CTL) response. This results in cell lysis and ultimately the death of the infected cell [ 161. However, sequencing of individual pep- tides presents a substantial challenge since many of these peptides are present at the nanomolar-picomolar levels [17]. In order to overcome this problem, it is important to minimize analyte loss and maximize MS and tandem MS sensitivity. In that regard, we have inter- faced mPC-CE with a purpose-built, sheathless micros- pray ionization source (mPC-CE-pMS) (see Fig. 2) [18], based on a liquid junction microspray design originally reported by Yates 1191. We demonstrate the utility of this approach in the sequence analysis of antigenic MHC class I peptides derived from -lo9 EG-7 mouse tumor cells. 2 Materials and methods 2.1 Chemicals and materials Acetic acid (99% + grade), Polybrene and ethylene glycol were obtained from Aldrich (Milwaukee, WI, USA), and ammonium acetate (99.9% grade), HPLC-grade meth- anol, and water from Baxter (Minneapolis, MN, USA). The standard peptide mixture containing bradykinin, bradykinin 1-5, IArg*]-vasopressin, leucine-enkephalin, 0 WILEY-VCH Verlag GmbH, 69451 Weinheim, 1998 0173-0835/98/1212-2207 $17.50+.50/0