Accelerated proteolytic cleavage of proteins under controlled microwave irradiation has been achieved. Selective peptide fragmentation by endoproteases trypsin or lysine C led to smaller peptides that were analyzed by matrix-assisted laser desorption ionization (MALDI) or liquid chromatography-electrospray ionization (LC-ESI) techniques. The efficacy of this technique for protein mapping was demonstrated by the mass spectral analyses of the peptide fragmentation of several biologically active proteins, including cytochrome c, ubiquitin, lysozyme, myoglobin, and interferon ␣-2b. Most important, using this novel approach digestion of proteins occurs in minutes, in contrast to the hours required by conventional methods.

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The capability for accurate mass measurements is an important attribute of Fourier transform mass spectrometry (FTMS). Unlike other instrumental methods in mass spectrometry, FTMS still offers significant opportunities to improve mass measurement accuracy (MMA), making it an area of research. This review covers the published literature in FTMS from the late 1970s to the present. We discuss the development and evolution of mass calibration that give accuracies in the low ppm range. We sketch the derivation and show the common foundation of these mass calibration procedures. We also describe the relation of mass calibration and the fundamentals of ion motion and space-charge effects, and we review efforts to improve the basic calibration procedure particularly those that correct for effects of space charge. The advent of matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) have opened the door for FTMS to be used for analyzing at high performance biopolymers, including proteins, oligodeoxynucleotides (ODNs), oligosaccharides, and synthetic polymers. We also discuss the utility of FTMS for accurate mass measurement in these areas and some practical ways to improve MMA. # 2004 Wiley Periodicals, Inc., Mass Spec Rev 24:286-309, 2005

SutnlmaryWe and others have previously reported that melanoma-specific, cytotoxic T lymphocytes (CTL) define a minimum of six class I-presented peptide epitopes common to most HLA-A2 + melanomas. Here we show that three of these peptide epitopes are coordinately recognized by a CTL clone obtained by limiting dilution from the peripheral blood of an HLA-A2 + melanoma patient. Tandem mass spectrometry was used to characterize and sequence one of these three naturally processed melanoma peptides. One of the potential forms of the deduced peptide sequence (XXTVXXGVX, X = I or L) matches positions 32-40 of the recently identified melanoma gene MART-1/Melan-A. This peptide (p939; ILTVILGVL) binds to HLA-A2 with an intermediateto-low affinity and is capable of sensitizing the HLA-A2 § T2 cell line to lysis by CTL lines and clones derived from five different melanoma patients. A relative high frequency of anti-p939-specific effector cells appear to be present in situ in HLA-A2 + melanoma patients, since p939 is also recognized by freshly isolated tumor infiltrating lymphocytes, p939 represents a good candidate for the development of peptide-based immunotherapies for the treatment of patients with melanoma.