Proteomic analysis of platelet α-granules using mass spectrometry
Article first published online: 10 JUL 2007
© 2007 International Society on Thrombosis and Haemostasis
Journal of Thrombosis and Haemostasis
Volume 5, Issue 9, pages 1945–1955, September 2007
How to Cite
MAYNARD, D. M., HEIJNEN, H. F. G., HORNE, M. K., WHITE, J. G. and GAHL, W. A. (2007), Proteomic analysis of platelet α-granules using mass spectrometry. Journal of Thrombosis and Haemostasis, 5: 1945–1955. doi: 10.1111/j.1538-7836.2007.02690.x
- Issue published online: 10 JUL 2007
- Article first published online: 10 JUL 2007
- Received 24 August 2006, accepted 29 June 2007
Table S1. Proteins identified in fraction 6 gel experiments using LC-MS/MS (n = 3). Proteins listed must meet the minimum criteria for inclusion in the table. Each peptide used for a protein identification must have a Mascot ion score that meets or exceeds its identitiy score. Each protein listed must have a minimum of two peptides from at least 2 out of 3 gel experiments. Mitochondrial proteins have been removed from the list.
Fig S1. A. Light microscopy of whole platelets obtained after washing but before lysis by ultrasonication. Stained with Wright-Giemsa stain; size bar = 5 µm. B. Transmission electron microscopy of whole platelets obtained after washing but before lysis by ultrasonication. Note the presence of intact alpha granules and the relative absence of pseudofilia, reflecting lack of platelet activation. Scope magnification x 33,000.
Fig S2. A. Picture of the sucrose gradient following ultracentrifugation. Fractions were collected by pipetting from the top. Fractions 2 and 4 were saved and prepared for immuno-electron microscopy. B?G. Immuno-electron micrographs of fractions 2 (B?D) and 4 (E?G) using three different antibody labels. B and E: Anti-Glut-3; C and F: Anti-CD63; D and G: Anti-GPIb. Fraction 2 contained high amounts of membranes that were positive for the plasma membrane (PM) and open canalicular system (OCS) marker Ak3 (the antibody for GPIBα) (Fig. D), whereas Glut-3 was found on only a few vesicular membranes (Fig. B). Fraction 2 also contained the highest amount of CD63-positive membranes (Fig. C). Fraction 4 contained high numbers of dense tubular membranes,possibly related to the platelet dense tubular system (DTS); these membranes are negative for both CD63 and GPIb. However, a population of vesicles and sheets of fraction 4 were positive for CD63 and GPIb (Fig. F and G, respectively). Occasional labeling of vesicles with Glut-3 was also found in fraction 4 (Fig. E). Size bar = 200 nM (B, C, E, F,G); 100 nM (D). H. SDS-Page gel of fractions 2 and 4 from the sucrose gradient. Each lane was loaded with 40 µg total protein and subsequently stained with Coomassie Brilliant Blue R-250 Staining Solution. The gels were sliced from top to bottom,then reduced, alkylated, and digested with trypsin. Resulting peptides were detected by LC-MS/MS.
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