Pox proteomics: mass spectrometry analysis and identification of Vaccinia virion proteins

Abstract

Background: Although many vaccinia virus proteins have been identified and studied in detail, only a few studies have attempted a comprehensive survey of the protein composition of the vaccinia virion. These projects have identified the major proteins of the vaccinia virion, but little has been accomplished to identify the unknown or less abundant proteins. Obtaining a detailed knowledge of the viral proteome of vaccinia virus will be important for advancing our understanding of orthopoxvirus biology, and should facilitate the development of effective antiviral drugs and formulation of vaccines.

Results: In order to accomplish this task, purified vaccinia virions were fractionated into a soluble protein enriched fraction (membrane proteins and lateral bodies) and an insoluble protein enriched fraction (virion cores). Each of these fractions was subjected to further fractionation by either sodium dodecyl sulfate-polyacrylamide gel electophoresis, or by reverse phase high performance liquid chromatography. The soluble and insoluble fractions were also analyzed directly with no further separation. The samples were prepared for mass spectrometry analysis by digestion with trypsin. Tryptic digests were analyzed by using either a matrix assisted laser desorption ionization time of flight tandem mass spectrometer, a quadrupole ion trap mass spectrometer, or a quadrupole-time of flight mass spectrometer (the latter two instruments were equipped with electrospray ionization sources). Proteins were identified by searching uninterpreted tandem mass spectra against a vaccinia virus protein database created by our lab and a non-redundant protein database.

Conclusion: Sixty three vaccinia proteins were identified in the virion particle. The total number of peptides found for each protein ranged from 1 to 62, and the sequence coverage of the proteins ranged from 8.2% to 94.9%. Interestingly, two vaccinia open reading frames were confirmed as being expressed as novel proteins: E6R and L3L.

Figure 1

Mass analysis of a distinct peptide from the L4R protein using Method 1 (SDS-PAGE + LC-ESI-Q-TOF MS) Panel A shows the Coomassie blue stained SDS-PAGE gel of the core-enriched fraction and panel B is the membrane-enriched fraction. Gel slices that were analyzed by MS are denoted with letters. The full scan mass spectrum (inset of C) displays a doubly charged parent ion at m/z 867.9. The corresponding tandem mass spectrum (C) identifies a peptide of the L4R protein. Asterisks (*) denote the loss of ammonia (NH₃) or water (H₂O).

Figure 2

Mass analysis of a distinct peptide from the E6R protein using Method 2 (SDS-PAGE + LC-ESI-QIT MS) Gel slice "d" from the SDS-PAGE of the core-enriched fraction (Fig. 1A) was subjected to an in-gel trypsin digestion, and analyzed by LC-ESI-QIT MS. The tandem mass spectrum data, correlating to the full scan mass spectrum (inset, doubly charged parent ion at m/z 831.1), reveals a peptide of the E6R protein. Asterisks (*) denote the loss of ammonia (NH₃) or water (H₂O).

Figure 3

Mass analysis of a distinct peptide from the L1R protein using Method 3 (HPLC + LC-ESI-QIT MS). The core- (A) and membrane-enriched (B) fractions were resolved on a C₄ HPLC column according to the Methods section. Tandem mass spectrometric analysis of fraction 59–60 (B, indicated by brackets) produced from a singly charged precursor ion (inset, m/z 1289.7), yielded fragment ions which corresponded to a peptide the L1R protein. Asterisks (*) denote the loss of ammonia (NH₃) or water (H₂O).

Figure 4

Mass analysis of a distinct peptide from the L3L protein using Method 4 (LC-ESI-Q-TOF MS). The core-enriched fraction of the virion was subjected to trypsin digestion, and analyzed by the LC-ESI-Q-TOF mass spectrometer. The full scan mass spectrum displays a peak at m/z 844.5 (inset), and corresponding tandem mass spectrum identifies a peptide of the L3L protein. Four internal fragments were also identified for the L3L peptide including: PL, GFP, PLL, and GFPL.

Figure 5

Mass analysis of a distinct peptide from the G3L protein using Method 5 (MALDI-TOF/TOF). The membrane-enriched fraction of the virion was subjected to trypsin digest, and analyzed by MALDI-TOF/TOF MS. The full scan mass spectrum yielded a singly charged ion at m/z 1522.69 (inset). Tandem mass spectrum of the parent ion corresponds to a peptide of G3L. Asterisks (*) denote the loss of ammonia (NH₃) or water (H₂O).

Figure 6

Diagram of proteins identified using multiple methods. All overlaps are shown for all five methods: Method 1 - SDS-PAGE + LC-ESI-Q-TOF MS (dashed line); Method 2 - SDS-PAGE + LC-ESI-QIT MS (double line); Method 3 -HPLC + LC-ESI-QIT MS (solid line); Method 4 - LC-ESI-Q-TOF MS (dotted line); and Method 5 - MALDI-TOF/TOF (triple line). Numbers represent the number of shared proteins in overlapping areas. The total number of proteins identified for the method and the percent of total proteins identified is listed.

Figure 7

Summary of the functions of the 63 identified VV proteins. This graph summarizes the 63 VV proteins identified in this study according to their function. These include: core structural proteins (A3L, A4L, A10L, A12L, A14L, and L4R), membrane proteins (A13L, A17L, A27L, A33R, A34R, A56R, B5R, D8L, F9L, F13L, H3L, H5R, J1R, and L1R), transcriptional proteins (A5R, A7L, A24R, A29L, D1R, D6R, D11L, D12L, E1L, E3L, E4L, H4L, I1L, I8R, J3R, J4R, J6R, and K4L), proteins with other functions (A15L, A16L, A30L, A42R, A46R, B22R, D2L, D3R, E8R, E10R, E11L, F8L, F10L, F17R, G1L, G3L, G4L, G7L, H1L, I3L, I5L, I7L, and O2L), and the unknown proteins (E6R and L3L).