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.

An ion trap source has been designed for use with time-of-flight (TOF) mass analysis. Two thin diaphragms make up a segmented ring electrode; the end cap electrodes are planar wire mesh. The potential field produced by the rf voltage applied between the ring and end cap electrodes resembles that of the cylindrical ion trap. The trapped ion population for ions created by electron impact exhibits linear growth against a first-order loss that has a time constant of about 50 µs; no ion loss occurs when the electron beam is off. The observed value of q z at low-mass cutoff for rf ion storage is -0.84. Pulsed extraction of all ions is accomplished by switching the trap electrodes from rf to voltages required to provide a linear dc extraction field. The TOF flight path includes a wide energy range reflectron. Better than unit mass resolution is achieved through m/z 500 without collisional ion cooling. With an extraction rate of 1 kHz and a recording rate of 4 spectra per second, a linear working curve is obtained between 36 pg and 18 ng of chlorobenzene delivered chromatographically. The system has demonstrated the potential to achieve a very high sample utilization efficiency at high spectral generation rates.

A novel method for the design of reflections capable of focusing large kinetic energy ranges is presented. The design method itself is a numerical approach that provides a geometrically flexible alternative to traditional analytical design solutions. This design method has been used to produce a reflectron that provides unit mass resolution for product spectra in a tandem reflectron time-of-flight (TOF) mass spectrometer despite a kinetic energy range of 1950-2700 eV. In this application, the systematic progression of reflectron design results in a practical, nonlinear field reflectron with the use of only two grids. Design improvements are proposed for more flexible systems, although geometric constraints in the current instrument limit their experimental evaluation.