Highly active cytochrome b 6 f complexes from spinach and the cyanobacterium Mastigocladus laminosus have been analyzed by liquid chromatography with electrospray ionization mass spectrometry (LCMS؉). Both sizeexclusion and reverse-phase separations were used to separate protein subunits allowing measurement of their molecular masses to an accuracy exceeding 0.01% (؎3 Da at 30,000 Da). The products of petA, petB, petC, petD, petG, petL, petM, and petN were detected in complexes from both spinach and M. laminosus, while the spinach complex also contained ferredoxin-NADP

Aconitases are iron-sulfur cluster-containing proteins present both in mitochondria and cytosol of cells; the cubane iron-sulfur (Fe-S) cluster in the active site is essential for catalytic activity, but it also renders aconitase highly vulnerable to reactive oxygen and nitrogen species. This study examined the sites and mechanisms of aconitase inactivation by peroxynitrite (ONOO-), a strong oxidant and nitrating agent readily formed from superoxide anion and nitric oxide generated by mitochondria. ONOO- inactivated aconitase in a dose-dependent manner (half-maximal inhibition was observed with approximately 3 microM ONOO-). Low levels of ONOO- caused the conversion of the Fe-S cluster from the [4Fe-4S]2+ form to the inactive [3Fe-4S]1+ form with the loss of labile iron, as confirmed by low-temperature EPR analysis. In the presence of the substrate, citrate, 66-fold higher concentrations of ONOO- were required for half-maximal inhibition. The protective effects of citrate corresponded to its binding to the active site. The inactivation of aconitase in the presence of citrate was due to ONOO--mediated cysteine thiol loss and tyrosine nitration in the enzyme as shown by Western blot analyses. LC/MS/MS analyses revealed that ONOO- treatment to aconitase resulted in nitration of tyrosines 151 and 472 and oxidation to sulfonic acid of cysteines 126 and 385. The latter is one of the three cysteine residues in aconitase that binds to the Fe-S cluster. All other modified tyrosine and cysteine residues were adjacent to the binding site, thus suggesting that these modifications caused conformational changes leading to active-site disruption. Aconitase cysteine thiol modifications other than oxidation to sulfonic acid, such as S-glutathionylation, also decreased aconitase activity, thus indicating that glutathionylation may be an important means of modulating aconitase activity under oxidative and nitrative stress. Taken together, these results demonstrate that the Fe-S cluster in the active site, cysteine 385 bound to the Fe-S cluster, and tyrosine and cysteine residues in the vicinity of the active site are important targets of oxidative and/or nitrative attack, which is selectively controlled by the mitochondrial matrix citrate levels. The mechanisms inherent in aconitase inactivation by ONOO- are discussed in terms of the mitochondrial matrix metabolic and thiol redox state.

The numbers of host-adapted Borrelia burgdorferi (HAB) organisms in rabbit skin were assessed by real-time PCR over the first 3 weeks of infection. Maximal numbers were found at day 11, while spirochete numbers decreased by more than 30-fold by day 21. The antigenic composition of HAB in skin biopsy samples was determined by use of a procedure termed hydrophobic antigen tissue Triton extraction. Immune sera from rabbits, sera from chronically infected mice, and monospecific antiserum to the antigenic variation protein, VlsE, were used to probe parallel two-dimensional immunoblots representing each time point. Individual proteins were identified using either specific antisera or by matching protein spots to mass spectrometryidentified protein spots from in vitro-cultivated Borrelia. There were significant changes in the relative expression of a variety of known and previously unrecognized HAB antigens during the 21-day period. OspC and the outer membrane proteins OspA and OspB were prominent at the earliest time point, day 5, when the antigenic variation protein VlsE was barely detected. OspA and OspB were not detected after day 5. OspC was not detected after day 9. VlsE was the most prominent antigen from day 7 through day 21. BmpA, ErpN, ErpP, LA7, OppA-2, DbpA, and an unidentified 15-kDa protein were also detected from day 7 through day 21. Immunoblot analysis using monospecific anti-VlsE revealed the presence of prominent distinct VlsE lower forms in HAB at days 9, 11, and 14; however, these lower forms were no longer detected at day 21. This marked diminution in VlsE lower forms paralleled the clearance of the spirochete from skin.

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.

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We aimed to gain further understanding of the molecular mechanisms involved in oxaliplatin resistance in colorectal cancer by using a proteomic approach. A 5-fold oxaliplatinresistant cell line, HTOXAR3, was compared with its parental cell line, HT29, using two-dimensional PAGE. Mass spectrometry, Western blot, and real-time quantitative PCR confirmed the down-regulation of pyruvate kinase M2 (PK-M2) in HTOXAR3 cells. In a panel of eight colorectal cancer cell lines, we found a negative correlation between oxaliplatin resistance and PK-M2 mRNA levels (Spearman r = −0.846, P = 0.008). Oxaliplatin exposure in both HT29 and HTOXAR3 led to PK-M2 mRNA up-regulation. PK-M2 mRNA levels were measured by real-time quantitative PCR in 41 tumors treated with oxaliplatin/5-fluorouracil. Tumors with the lowest PK-M2 levels attained the lowest response rates (20% versus 64.5%, P = 0.026). High PK-M2 levels were associated with high p53 levels (P = 0.032). In conclusion, the data provided clearly link PK-M2 expression and oxaliplatin resistance mechanisms and further implicate PK-M2 as a predictive marker of response in patients with oxaliplatintreated colorectal cancer. [Mol Cancer Ther 2009;8(4): 771-8]

Background: Carbon isotope ratio methods are used in doping control to determine whether urinary steroids are endogenous or pharmaceutical. Methods: Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) was used to determine the δ13C values for 5β-androstane-3α,17β-diyl diacetate (5βA), 5α-androstane-3α,17β-diyl diacetate (5αA), and 5β-pregnane-3α,20α-diyl diacetate (5βP) in a control group of 73 healthy males and 6 athletes with testosterone/epitestosterone ratios (T/E) >6. Results: The within-assay precision SDs for 5βA, 5αA, and 5βP were ± 0.27‰, ± 0.38‰, and ± 0.28‰, respectively. The between-assay precision SDs ranged from ± 0.40‰ to ± 0.52‰. The system suitability and batch acceptance scheme is based on SDs. For the control group, the mean δ13C (SD) values were −25.69‰ (± 0.92‰), −26.35‰ (± 0.68‰), and −24.26‰ (± 0.70‰), for 5βA, 5αA, and 5βP, respectively. 5βP was greater than 5βA and 5αA (P <0.01), and 5βA was greater than 5αA (P <0.01). The means − 3 SD were −28.46‰, −28.39‰, and −26.37‰ for 5βA, 5αA, and 5βP, respectively. The maximum difference between 5βP and 5βA was 3.2‰, and the maximum 5βA/5βP was 1.13. Three athletes with chronically elevated T/Es had δ13C values consistent with testosterone administration and three did not. Conclusions: This GC-C-IRMS assay of urine diols has low within- and between-assay SDs; therefore, analysis of one urine sample suffices for doping control. The means, SDs, ±3 SDs, and ranges of δ13C values in a control group are established. In comparison, testosterone users have low 5βA and 5αA, large differences between 5βA or 5αA and 5βP, and high 5βA/5βP and 5αA/5βP ratios.