Antigens recognized by T cells are expressed as peptides bound to major histocompatibility complex (MHC) molecules. Microcapillary high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry was used to fractionate and sequence subpicomolar amounts of peptides isolated from the MHC molecule HLA-A2.1. Of 200 different species quantitated, eight were sequenced and four were found in cellular proteins. All were nine residues long and shared a distinct structural motif. The sensitivity and speed of this approach should enhance the analysis of peptides from small quantities of virally infected and transformed cells as well as those associated with autoimmune disease states.* Contributions of these authors were equivalent and their order should be considered arbitrary. HHS Public AccessAuthor manuscript J Immunol. Author manuscript; available in PMC 2015 October 28. Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptCytotoxic T lymphocytes (CTLs) are a part of the immune system concerned with recognition of host cells that express new antigens as a result of viral infection or transformation. CTLs do not recognize new antigens directly, but only as short peptides bound to a deep cleft in class I molecules of the MHC (1-3). Newly synthesized viral and cellular proteins are degraded into peptides in the cytoplasm, transported to the endoplasmic reticulum where they bind to class I molecules, and then expressed on the cell surface (4-7). Each of the allelic forms of the class I MHC molecule binds to a complex mixture of structurally distinct peptides (8,9). Information on the nature of these peptides has been obtained from studies with synthetic peptides (10-12) and from Edman degradation applied to unfractionated mixtures of peptides extracted from five different class I MHC molecules (8). Sequences of 11 peptides extracted from HLA-B27 were identified after highperformance liquid chromatography (HPLC) fractionation and Edman degradation (9). Because HPLC was unable to completely resolve the complex mixture, this analysis could only be applied to the few fractions that contained one or two dominant peptides. Declining PTH (phenylthiohydantoin)-amino acid yields made it difficult to determine the exact number of residues in several peptides.We have applied microcapillary HPLC-electrospray ionization-tandem mass spectrometry to circumvent the above problems. In a matter of hours, this technique determines the molecular mass and therefore maximum length of each peptide component, and the approximate number and quantity of individual peptides. Sequence information can be also obtained on subpicomolar amounts of peptides. We analyzed the naturally processed peptides bound to HLA-A2.1, one of the most widely distributed class I molecules within the human population. The three-dimensional structure of this molecule allows modeling of the complex (2).HLA-A2.1 molecules were purified by immunoprecipitation from the human B lymphoblastoid cell line C1R-A2.1. The associated p...

Between 650 and 2000 different peptides are associated with the major histocompatibility complex class II molecule I-Ad. Sequences for nine of these were obtained by a combination of automated Edman degradation and tandem mass spectrometry. All of the peptides are derived from secretory or integral membrane proteins that are synthesized by the antigen-presenting cell itself. Peptides were 16 to 18 residues long, had ragged NH2-and COOH-termini, and contained a six-residue binding motif that was variably placed within the peptide chain. Binding data on truncated peptides suggest that the peptide binding groove on class II molecules can be open at both ends.

Peptides extracted from HLA-A2.1 class I major histocompatibility complex (MHC) molecules expressed on the antigen processing mutant CEMx721.174.T2 were characterized by electrospray ionization-tandem mass spectrometry. Only seven dominant peptides were found, in contrast to over 200 associated with HLA-A2.1 on normal cells. These peptides were derived from the signal peptide domains of normal cellular proteins, were usually larger than nine residues, and were also associated with HLA-A2.1 in normal cells. These results suggest that proteolysis of signal peptide domains in the endoplasmic reticulum is a second mechanism for processing and presentation of peptides for association with class I molecules.

Class III fi-tubulin, isolated from adult bovine brain, is resolved into at least seven charge variants on isoelectric focusing gels. To identify the posttranslational modifications responsible for this heterogeneity, a mixture of brain tubulins was treated with cyanogen bromide and the C-terminal fragments from the class III I3-tubulin isoforms were then isolated by binding them to the monoclonal antibody TuJ1. Combined use of tandem mass spectrometry and both subtractive and automated Edman degradation chemistry on the isolated peptides indicates that many of the isoforms differ by phosphorylation at Ser-444 plus attachment of one to six glutamic acid molecules to the side chain of the rst glutamate residue, Glu-438, in the C-terminal sequence Tyr-Glu-AspAsp-Glu-Glu-Glu-Ser-Glu-Ala-Gln-Gly-Pro-Lys.Microtubules, assembled from two similar 50-kDa proteins designated a-and f3-tubulin, are involved in a number of important biological processes including segregation of chromosomes during cell division, cell motility, organelle transport, and maintenance of cell shape (1). Brain tubulin exhibits a high degree of heterogeneity. Up to 21 charge variants have been observed by isoelectric focusing (IEF) techniques (2-4).Both a-and 83-tubulins are encoded by small multigene families (5). Since the number of charge variants exceeds the number of tubulin genes by approximately a factor of 2, the remaining isoforms are assumed to result from posttranslational modifications. a-Tubulin is reported to undergo acetylation (6-8), glutamylation (9), and the addition and removal of a C-terminal tyrosine residue (10, 11). Phosphorylation of a single seine residue in one of the five P-tubulin isotypes expressed in brain has also been described (12-16).Class III p8-tubulin, a vertebrate isotype found only in neurons and cells in the mammalian testis (17), undergoes a developmentally regulated increase in heterogeneity in the former but not the latter tissue (18,19). The testis and earliest embryonic rat brain isoforms cofocus on an IEF gel (19). At least six additional isoforms of neuron-specific class III ,B-tubulin are detected in the adult rat or bovine brain.Previous work localized the site of heterogeneity to the C-terminal 20 amino acids (19), a highly acidic region of the protein thought to be involved in the interaction of microtubules with both calcium ions (20) and microtubule-associated proteins (21-23).To further characterize both the nature and location of structural modifications responsible for the observed heterogeneity, class III /3-tubulin from bovine brain was subjected to amino acid sequence analysis by tandem mass spectrometry. We report that the neuron-specific class III /3-tubulin isoforms result in part from phosphorylation at Ser-444 and attachment of at least three and perhaps as many as six glutamic acid molecules to the side chain of the first glutamate residue, Glu-438, in the C-terminal sequence Tyr-GluAsp-Asp-Glu-Glu-Glu-Ser-Glu-Ala-Gln-Gly-Pro-Lys (YED-DEEESEAQGPK). MATERIALS AND METHODSIsolati...

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