Accurate mass precursor ion data and tandem mass spectrometry identify a class I human leukocyte antigen A*0201-presented peptide originating from vaccinia virus

Abstract

We have used accurate mass precursor ion data generated on a hybrid linear-ion trap-Fourier transform ion cyclotron resonance mass spectrometer to augment tandem mass spectrometry (MS/MS) data generated on two different instrument types. Results from these experiments have allowed us for the first time to identify a naturally processed peptide presented by a class I human leukocyte antigen allele (HLA-A*0201) that was isolated from B cells infected by live vaccinia, the viral agent of the smallpox vaccine. The accurate mass data, in conjunction with MS/MS data, was able to identify the sequence IVIEAIHTV (aa 187-195) from the protein thymidylate kinase of vaccinia, distinguishing it from a similar sequence IVLEAIAEH: a "self-peptide" from the human protein phospholipase C␤3. Accurate mass data for the doubly charged species from the naturally processed and presented peptide was 497.8006, which was within 0.8 ppm of the calculated m/z of 497.8002, while being Ϫ37.3 ppm from the calculated m/z (497.7820) of the second-ranked peptide sequence IVLEAIAEH. Accurate mass data ranged from less than 0.1 to 1.2 ppm for other peptides identified in this sample. A BLAST search shows this sequence, IVIEAIHTV, is conserved in the same protein of a number of other orthopoxviruses, including the variola (smallpox) virus. Additionally, accurate mass data were able to uncover a false positive search result that was not distinguished by scoring of the match to the MS/MS data. [5,6] have successively enabled other researchers in the application of mass spectrometry to their respective areas of research. As an example, we have employed the use of multidimensional LC together with automated acquisition of MS/MS data for the identification of selfpeptides and peptides originating from measles virus proteins that were displayed by the human leukocyte antigen (HLA) class II system from cultured B cells infected with measles virus [7][8][9].Recently, the development and commercialization of hybrid mass spectrometers incorporating Fourier transform ion cyclotron resonance (FTICR) analyzers, with high resolving power (RP) and high mass accuracy offer additional opportunities to enhance the usefulness of

Long-Term Immunity against Actual Poxviral HLA Ligands as Identified by Differential Stable Isotope Labeling

“…IL-4 and IL-7 (5 ng/ml; R&D Systems) were added after thawing. On day 1, peptides (1 g/ml) prepared as mixtures of five peptides each were added to PBMC, as follows: mix 1, F12L 404 -412 , G5.5R [27][28][29][30][31][32][33][34][35] [303][304][305][306][307][308][309][310][311] , and B15R 91-101 ; and IL-4 and IL-7 (5 ng/ml) were added. On days 3, 5, and 7, IL-2 (2 ng/ml; R&D Systems) was added.…”

“…revealed specificity for seven of the identified HLA-A*0201 ligands (Table V). All three donors contained CD8 ϩ T cells specific for two of the common epitopes identified in the previous experiments with MVA vaccinees, C7L 74 -82 and F12L 404 -412 , whereas two donors contained T cells specific for G5.5R [27][28][29][30][31][32][33][34][35] and O1L 247-255 . These results suggest that one round of prime-boost vaccination with MVA or Dryvax was sufficient to induce a long-lived cellular immune response to several identical epitopes.…”

“…In the mock-immunized group, all mice were dead by day 7; one mouse in the control and one mouse in the late group also died on day 7 (n ϭ 5). G5.5R [27][28][29][30][31][32][33][34][35] , and C7L 74 -82 ) (Fig. 6B).…”

“…Thus, directly sequenced HLA ligands have been considered to be more reliable than determinants identified solely by T cell analysis (30), which is mandatory to accurately determine the magnitude of specific CD8 ϩ T cell responses or the functionality of the CD8 ϩ T cells that respond to an epitope. To date, only one MVA-derived HLA ligand has been identified by MS, however, without demonstrating immunogenicity (31). Ideally, immunologically relevant viral T cell epitopes are confirmed both as HLA ligands and as T cell stimulators.…”

Structural characterization of monohydroxyeicosatetraenoic acids and dihydroxy- and trihydroxyeicosatrienoic acids by ESI-FTICR

“…M ass spectrometry has emerged as an important tool for analysis of biomolecules. Fourier transform ion cyclotron resonance (FTICR) mass spectrometry is one of the techniques that can provide high mass accuracy and high mass resolution [1][2][3][4][5][6]. Accurate molecular weight, elemental composition, and structural information can be achieved from FTICR.…”