Blonde

Abstract

The beneficial effects on peak selectivity and resolution of conducting liquid chromatography (LC) at elevated temperature (e.g., 30-80 °C) are generally well-known; however, its importance for peptide recovery is not nearly as well recognized. This report demonstrates that µLC analysis of membrane proteomic samples significantly benefits from the application of heat. Enriched membrane and membrane-embedded peptides (the latter obtained by membrane shaving) were analyzed by µLC-tandem mass spectrometry (MS/MS) from 20 to 60 °C using a standard reversed-phase material. Maximal protein and hydrophobic peptide recovery was obtained at 60 °C. The membrane-shaving method employed, a recently optimized version of the high pH/proteinase K protocol, provided significant integral membrane protein enrichment: 98% of identified proteins were predicted to have at least one transmembrane domain (87% to have at least three), and 68% of peptides were predicted to contain transmembrane segments. Analysis of this highly enriched sample at elevated temperature increased protein identifications by 400%, and peptide identifications by 500%, as compared to room-temperature separation. Given that most µLC-MS/MS analyses are currently conducted at room temperature, the findings described herein should be of considerable value for improving the comprehensive study of integral membrane proteins. Integral membrane proteins (IMPs) are critical for the maintenance of biological systems and represent important targets for the treatment of disease. To have a more complete understanding of their myriad roles in patho/physiological processes, detailed proteomic information [e.g., abundance, activity, localization, structure, sequence, post-translational modifications (PTMs), and molecular interactions] is required. However, given the challenging nature of these amphipathic species, even basic characterization of IMPs lags far behind that of their soluble counterparts. For many years, the gold standard for complex proteome analysis was 2D gel electrophoresis followed by identification using mass spectrometry; however, even with recent improvements, most IMP classes remain resistant to characterization. 1 In response to the difficulties inherent to gel-based separation of intact IMPs, shotgun 2 proteomic characterization was adopted. 3-5 Shotgun proteomics combines chromatographic, electrophoretic, or affinity purification separation of complex protein digests with electrospray ionization (ESI) 6 or matrix-assisted laser desorption/ionization (MALDI), 7 commonly in the form of microcapillary liquid chromatography tandem mass spectrometry (µLC-MS/MS). 8 However, sample preparation still presented a problem for IMPs due to their intractable solubilization in ESI-compatible detergents and subsequent incomplete digestion. 9 In response, several creative solutions were introduced, including digestion in aqueous methanol, 4,10-13 proteolytic shaving of soluble domains, 12-16 and biotin-10,17 or lectin-mediated 18-22 affinity enrichment. As will be discussed, however, because shotgun strategies were originally optimized for soluble peptides, typical µLC separation strategies may also require Goshe, M. B.; Moore, R. J.; Pasa-Tolic, L.; Masselon, C. D.; Lipton, M. S.; Smith, R. D. J. Proteome Res. 2002, 1, 351-360. (5) Blonder, J.; Terunuma, A.; Conrads, T. P.; Chan, K. C.; Yee, C.; Lucas, D. A.; Schaefer, C. F.; Yu, L. R.; Issaq, H. J.; Veenstra, T. D.; Vogel, J. C. Conrads. T. P.; Yu, L.-R.; Terunuma, A.; Janini, G. M.; Issaq, H. J.; Vogel, J. C.; Veenstra, T. D. Proteomics 2004, 4, 31-35. (12) Fischer, F.; Wolters, D.; Rogner, M.; Poetsch, A. Mol. Cell. Proteomics 2006, 5, 444-453. (13) Wei, C.; Yang, J.; Zhu, J.; Zhang, X.; Leng, W.; Wang, J.; Xue, Y.; Sun, L.; Li, W.; Wang, J.; Jin, Q. Norais, N.; Bensi, G.; Liberatori, S.; Capo, S.; Mora, M.; Scarselli, M.; Doro, F.; Ferrari, G.; Garaguso, I.; Maggi, T.; Neumann, A.; Covre, A.; Telford, J. L.; Grandi, G. Nat. Biotechnol. 2006, 24, 191-197. (15) Le Bihan, T.; Goh, T.; Stewart, I. I.; Salter, A. M.; Bukhman, Y. V.; Dharsee, M.; Ewing, R.; Wisniewski, J. R. Nagano, K.; Itagaki, C.; Taoka, M.; Okamura, N.; Yamauchi, Y.; Sugano, S.; Takahashi, N.; Izumi, T.; Isobe, T. Mol. Cell. Proteomics 2005, 4, 1968-1976. (18) Springer, D. L.; Auberry, D. L.; Ahram, M.; Adkins, J. N.; Feldhaus, J. M.; Wahl, J. H.; Wunschel, D. S.; Rodland, K. D. Dis. Markers 2004, 19, 219-228. (19) Ghosh, D.; Krokhin, O.; Antonovici, M.; Ens, W.; Standing, K. G.; Beavis, R. C.; Wilkins, J. A. J. Proteome. Res. 2004, 3, 841-850. (20) Fan, X.; She, Y. M.; Bagshaw, R. D.; Callahan, J. W.; Schachter, H.; Mahuran, D. J. Glycobiology 2005, 15, 952-964. (21) Zhang, H.; Li, X. J.; Martin, D. B.; Aebersold, R. Nat. Biotechnol. 2003, 21, 660-666. (22) Kaji, H.; Saito, H.; Yamauchi, Y.; Shinkawa, T.; Taoka, M.; Hirabayashi, J.; Kasai, K.; Takahashi, N.; Isobe, T. Nat. Biotechnol. 2003, 21, 667-672.