A new method for proteolytic stable isotope labeling is introduced to provide quantitative and concurrent comparisons between individual proteins from two entire proteome pools or their subfractions. Two 18O atoms are incorporated universally into the carboxyl termini of all tryptic peptides during the proteolytic cleavage of all proteins in the first pool. Proteins in the second pool are cleaved analogously with the carboxyl termini of the resulting peptides containing two 16O atoms (i.e., no labeling). The two peptide mixtures are pooled for fractionation and separation, and the masses and isotope ratios of each peptide pair (differing by 4 Da) are measured by high-resolution mass spectrometry. Short sequences and/or accurate mass measurements combined with proteomics software tools allow the peptides to be related to the precursor proteins from which they are derived. Relative signal intensities of paired peptides quantify the expression levels of their precursor proteins from proteome pools to be compared, using an equation described in the paper. Observation of individual (unpaired) peptides is mainly interpreted as differential modification or sequence variation for the protein from the respective proteome pool. The method is evaluated here in a comparison of virion proteins for two serotypes (Ad5 and Ad2) of adenovirus, taking advantage of information already available about protein sequences and concentrations. In general, proteolytic 18O labeling enables a shotgun approach for proteomic studies with quantitation capability and is proposed as a useful tool for comparative proteomic studies of very complex protein mixtures.

Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype.

No abstract

Proteolytic labeling in H2(18)O has been recently revived as a versatile method for proteomics research. To understand the molecular basis of the labeling process, we have dissected the process into two separate events: cleavage of the peptide amide bonds and exchange of the terminal carboxyl oxygens. It was demonstrated that both carboxyl oxygens can be catalytically labeled, independent of the cleavage step. Reaction kinetics of the tryptic 16O-to-18O exchange of YGGFMR, YGGFMK, and the tryptic digest of apomyoglobin were studied by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry. A larger KM for the Lys-peptide (4400 +/- 700 microM), when compared to that of the Arg-peptide (KM 1300 +/- 300 microM), was mainly responsible for the slower reaction with YGGFMK (kcat/KM 0.64 +/- 0.14 microM(-1)min(-1)) compared to YGGFMR (kcat/KM 2.6 +/- 0.9 microM(-1)min(-1)). Multiplexed kinetic studies showed that endoprotease-catalyzed oxygen exchange is a general phenomenon, allowing homogeneous 18O2-coding of a variety of peptides. It was demonstrated for the first time that chymotrypsin 18O2-codes peptides during proteolysis. On the basis of the analyses reported here, we propose that proteolytic 18O labeling can be advantageously decoupled from protein digestion, and endoproteases can be used in a separate step to 18O2-code peptides for comparative studies after proteolysis has taken place.

Myeloid-derived suppressor cells (MDSC) are a diverse population of immature myeloid cells that have potent immune-suppressive activity. Studies in both mice and humans have demonstrated that MDSC accumulate in most individuals with cancer, where they promote tumor progression, inhibit antitumor immunity, and are an obstacle to many cancer immunotherapies. As a result, there has been intense interest in understanding the mechanisms and in situ conditions that regulate and sustain MDSC, and the mechanisms MDSC use to promote tumor progression. This article reviews the characterization of MDSC and how they are distinguished from neutrophils, describes the suppressive mechanisms used by MDSC to mediate their effects, and explains the role of proinflammatory mediators and the tumor microenvironment in driving MDSC accumulation, suppressive potency, and survival.

Recently, proteolytic 18O labeling has been demonstrated as a promising strategy for comparative proteomic studies (Yao, X.; Freas, A.; Ramirez, J.; Demirev, P. A.; Fenselau, C. Anal. Chem. 2001, 73, 2836-42). In this approach, protein mixtures are digested in parallel in H216O and H218O and the ratios of isotopically distinct peptide products are measured by mass spectrometry. In the initial report from this laboratory, trypsin was shown to catalyze incorporation of two 18O atoms into the carboxyl terminus of each new peptide formed by cleavage of the adenovirus proteome. In the present study, a second enzyme, endoprotease Glu-C, is evaluated as an agent for cleavage and labeling. Proteolytic 18O labeling by Glu-C is shown to occur readily with phosphorylated and glycosylated proteins and with cysteinealkylated and disulfide-linked proteins. A sequential double-labeling strategy is used to characterize N-linked glycopeptides. Labeled and unlabeled peptide pairs are found to coelute chromatographically, and measurements of isotope ratios by nanospray and capillary LC-MS are found to be accurate and precise.

This study characterizes various features of the proteins that are detected in MALDI mass spectra when whole bacteria cells are analyzed, in an effort to understand why some proteins are successfully detected and many others are not. Forty peaks observed in the mass range 4,000-20,000 Da in the spectra of Escherichia coli K-12 and 11775 are tentatively assigned to proteins in a protein database, and these proteins are characterized by cell location, copy number, pI, and hydropathicity. Those detected originate in the cytosol and generally share the traits of high abundance within the cell, strong bacisity, and medium hydrophilicity.

A method for rapid identification of microorganisms is presented, which exploits the wealth of information contained in prokaryotic genome and protein sequence databases. The method is based on determining the masses of a set of ions by MALDI TOF mass spectrometry of intact or treated cells. Subsequent correlation of each ion in the set to a protein, along with the organismic source of the protein, is performed by searching an Internet-accessible protein database. Convoluting the lists for all ions and ranking the organisms corresponding to matched ions results in the identification of the microorganism. The method has been successfully demonstrated on B. subtilis and E. coli, two organisms with completely sequenced genomes. The method has been also tested for identification from mass spectra of mixtures of microorganisms, from spectra of an organism at different growth stages, and from spectra originating at other laboratories. Experimental factors such as MALDI matrix preparation, spectral reproducibility, contaminants, mass range, and measurement accuracy on the database search procedure are addressed too. The proposed method has several advantages over other MS methods for microorganism identification.