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Large-scale protein identifications from highly complex protein mixtures have recently been achieved using multidimensional liquid chromatography coupled with tandem mass spectrometry (LC/LC−MS/MS) and subsequent database searching with algorithms such as SEQUEST. Here, we describe a probability-based evaluation of false positive rates associated with peptide identifications from three different human proteome samples. Peptides from human plasma, human mammary epithelial cell (HMEC) lysate, and human hepatocyte (Huh)-7.5 cell lysate were separated by strong cation exchange (SCX) chromatography coupled offline with reversed-phase capillary LC−MS/MS analyses. The MS/MS spectra were first analyzed by SEQUEST, searching independently against both normal and sequence-reversed human protein databases, and the false positive rates of peptide identifications for the three proteome samples were then analyzed and compared. The observed false positive rates of peptide identifications for human plasma were significantly higher than those for the human cell lines when identical filtering criteria were used, suggesting that the false positive rates are significantly dependent on sample characteristics, particularly the number of proteins found within the detectable dynamic range. Two new sets of filtering criteria are proposed for human plasma and human cell lines, respectively, to provide an overall confidence of >95% for peptide identifications. The new criteria were compared, using a normalized elution time (NET) criterion (Petritis et al. Anal. Chem. 2003, 75, 1039−1048), with previously published criteria (Washburn et al. Nat. Biotechnol. 2001, 19, 242−247). The results demonstrate that the present criteria provide significantly higher levels of confidence for peptide identifications from mammalian proteomes without greatly decreasing the number of identifications. Keywords: proteomics • tandem mass spectrometry • the human proteome • LC−MS/MS • reversed database • SEQUEST criteria

We describe the application of a peptide retention time reversed phase liquid chromatography (RPLC) prediction model previously reported (Petritis et al. Anal. Chem. 2003, 75, 1039) for improved peptide identification. The model uses peptide sequence information to generate a theoretical (predicted) elution time that can be compared with the observed elution time. Using data from a set of known proteins, the retention time parameter was incorporated into a discriminant function for use with tandem mass spectrometry (MS/MS) data analyzed with the peptide/protein identification program SEQUEST. For singly charged ions, the number of confident identifications increased by 12% when the elution time metric is included compared to when mass spectral data is the sole source of information in the context of a Drosophila melanogaster database. A 3−4% improvement was obtained for doubly and triply charged ions for the same biological system. Application to the larger Rattus norvegicus (rat) and human proteome databases resulted in an 8−9% overall increase in the number of confident identifications, when both the discriminant function and elution time are used. The effect of adding “runner-up” hits (peptide matches that are not the highest scoring for a spectra) from SEQUEST is also explored, and we find that the number of confident identifications is further increased by 1% when these hits are also considered. Finally, application of the discriminant functions derived in this work with ~2.2 million spectra from over three hundred LC−MS/MS analyses of peptides from human plasma protein resulted in a 16% increase in confident peptide identifications (9022 vs 7779) using elution time information. Further improvements from the use of elution time information can be expected as both the experimental control of elution time reproducibility and the predictive capability are improved. Keywords: bioinformatics • proteome • algorithm • accurate mass and time tag • multivariate statistics • capillary liquid-chromatography • retention time • FTICR

Summary Rous sarcoma cells were implanted into the kidney of rats. After 5 days of growth the renal tumor was used for comparing histology with glucose 6-phosphatase (G6Pase) enzyme histochemistry (EHC) and18F-fluoro-2-deoxyglucose (FDG) auroradiography (ARG). It was found that the regions of the kidney tumor that had retained normal kidney structures were devoid of FDG, whereas there was histochemical staining of normal cortical areas. Regions of tumor growth, on the other hand, retained FDG and lacked G6Pase. Necrotic areas did not accumulate FDG. There was a dramatic decrease in the areas of G6Pase activity as a result of tumor infiltration in the kidney. The results show that FDG, currently being evaluated as a tumor detecting radiopharmaceutical indeed accumulates into areas of vital malignant growth, and they indicate that FDG positron emission tomographic (PET) images reveal the true anatomic location of malignant tissue.

Summary The combined and separate action of the antiestrogen toremifene (TOR) and recombinant rat gamma interferon (RIF) was studied in rat mammary cancer induced by dimethylbenzanthracene (DMBA). The content of ATP and14C-fluorodeoxyglucose (FDG) was also determined from fine needle aspiration biopsies (FNAB). RIF alone had no antitumor activity, when measured as the average number of new tumors appearing in RIF and control animals (2.4 vs 2.4 new tumors per animal), while TOR and TOR + RIF had a significant effect (1.2,P < 0.05 and 0.6,P < 0.01). Morphometrically, there was a significant decrease in the amount of epithelium in the tumors of the RIF + TOR animals (65% vs 82% in the controls,P < 0.05); there was conversely an increase in the stromal component (25% vs 14%, NS). It appears that an increase of the stromal compartment is part of the healing process. The feasibility of the FNAB-technique was shown by the finding that there was a close correlation between FDG and ATP content in almost all the groups before and after treatment. Thus, FDG and ATP measure the same phenomenon, i.e., energy content. There was a large variation in the contents of ATP and FDG within and among the groups, which invalidated considerations regarding the predictive value of ATP and FDG content in tumors subject to treatment.

Summary: The glucose analogue 2-fluoro-2-deoxy-d-glucose (FDG) was used to study chemosensitivity of two human ovarian cancer cell lines and of murine L1210 cells. Cell viability was determined by measuring intracellular adenosine triphosphate (ATP) with a bioluminescence method, which has been shown to correlate closely with trypan blue, stem cell, and [3H]TdR assays. All three cell lines were sensitive to cytostatic drugs, which exerted a parallel decrease in the intracellular FDG and ATP levels. The two measures correlated positively (r=0.66,P<0.001), indicating that FDG uptake is closely linked with ATP production. Relatively low hexokinase (HK)-to-glucose 6-phosphatase (HK/G6-Pase) ratios were measured, which suggests that the metabolic trapping of FDG 6-phosphate within the cytosol is incomplete. Apparently, these cell lines may not depend exclusively on glycolysis for their energy requirement. We conclude that cell killing caused by cytostatic drugs is associated with a decreased ATP content and FDG uptake. This indicates that not only ATP but also FDG may be used to study drug effects in vitro.