Purification and proteomic analysis of outer membrane vesicles from a clinical isolate of Leptospira interrogans serovar Copenhageni

• Published in: Proteomics (Weinheim) Vol. 5; no. 1; pp. 144 - 152
• Main Authors: Nally, Jarlath E., Whitelegge, Julian P., Aguilera, Rodrigo, Pereira, Martha M., Blanco, David R., Lovett, Michael A.
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 01.01.2005; WILEY‐VCH Verlag; Wiley-VCH
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Bacterial Outer Membrane Proteins - chemistry; Bacterial Outer Membrane Proteins - isolation & purification; Biological and medical sciences; Centrifugation, Density Gradient; Chromatography, High Pressure Liquid; Electrophoresis, Gel, Two-Dimensional; Fundamental and applied biological sciences. Psychology; Leptospira; Leptospira interrogans; Leptospira interrogans - chemistry; Liquid chromatography-electrospray ionization-mass spectrometry with fraction collection; Mass Spectrometry; Miscellaneous; Molecular Sequence Data; Outer membrane vesicles; Proteins; Proteomics; Two-dimensional gel electrophoresis; Purification; Spirochaetales; Leptospiraceae; Proteomics; Two-dimensional gel electrophoresis; Bacteria; Outer membrane vesicles; Leptospira interrogans; Liquid chromatography-electrospray ionization-mass spectrometry with fraction collection; External membrane; Clinical isolate; Leptospira
• ISSN: 1615-9853; 1615-9861
• DOI: 10.1002/pmic.200400880

The severe pulmonary form of leptospirosis (SPFL) is an especially serious and rapid disease process characterized by alveolar hemorrhage and acute respiratory failure. The outer membrane of Leptospira facilitates direct interactions with the environs and likely contains important constituents involved during infection, transmission, survival, and adaptation to environmental conditions, including putative vaccinogen and diagnostic candidates. Outer membrane vesicles (OMVs) were purified by incubation in low‐pH citrate buffer, treatment in a French press, and centrifugation over a continuous sucrose gradient. OMVs characterized by two‐dimensional gel electrophoresis (2‐DE) contained the previously described outer membrane proteins OmpL1, Qlp42, LipL32, LipL41, LipL36 and Loa22. In addition, unknown, hypothetical and putative outer membrane proteins were identified. High‐performance liquid chromatography (HPLC) coupled with mass spectrometry and fraction collection (LC‐MS+) measured the intact mass profile of the major outer membrane protein, LipL32, and the putative lipoprotein Qlp42. In contrast to a predicted molecularmass of 27 653.5 Da for LipL32 after cleavage of its signal peptide, intact mass proteomics measured the mass as ranging from 28 468 to 28 583 Da, consistent with lipidation of LipL32. In contrast to a predicted molecular mass of 39.8 kDa for Qlp42, the actual mass was measured as 24 811 and 26 461 Da consistent with a 30 kDa doublet observed on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gels and processing of the N‐terminus of the mature protein. These studies indicate that purified OMVs are highly compatible with proteomics technologies including 2‐DE and intact mass proteomics using LC‐MS+ that facilitates definition of actual molecular masses of intact outer membrane proteins, and heterogeneity associated with them.∁