A Multivariate Mixture Model to Estimate the Accuracy of Glycosaminoglycan Identifications Made by Tandem Mass Spectrometry (MS/MS) and Database Search.

We present a statistical model to estimate the accuracy of derivatized heparin and heparan sulfate (HS) glycosaminoglycan (GAG) assignments to tandem mass (MS/MS) spectra made by the first published database search application, GAG-ID. Employing a multivariate expectation-maximization algorithm, this statistical model distinguishes correct from ambiguous and incorrect database search results when computing the probability that heparin/HS GAG assignments to spectra are correct based upon database search scores. Using GAG-ID search results for spectra generated from a defined mixture of 21 synthesized tetrasaccharide sequences as well as seven spectra of longer defined oligosaccharides, we demonstrate that the computed probabilities are accurate and have high power to discriminate between correctly, ambiguously, and incorrectly assigned heparin/HS GAGs. This analysis makes it possible to filter large MS/MS database search results with predictable false identification error rates.

B-cell-independent sialylation of IgG.

IgG carrying terminal α2,6-linked sialic acids added to conserved N-glycans within the Fc domain by the sialyltransferase ST6Gal1 accounts for the anti-inflammatory effects of large-dose i.v. Ig (IVIg) in autoimmunity. Here, B-cell-specific ablation of ST6Gal1 in mice revealed that IgG sialylation can occur in the extracellular environment of the bloodstream independently of the B-cell secretory pathway. We also discovered that secreted ST6Gal1 is produced by cells lining central veins in the liver and that IgG sialylation is powered by serum-localized nucleotide sugar donor CMP-sialic acid that is at least partially derived from degranulating platelets. Thus, antibody-secreting cells do not exclusively control the sialylation-dependent anti-inflammatory function of IgG. Rather, IgG sialylation can be regulated by the liver and platelets through the corresponding release of enzyme and sugar donor into the cardiovascular circulation.

GAG-ID: Heparan Sulfate (HS) and Heparin Glycosaminoglycan High-Throughput Identification Software.

Heparin and heparan sulfate are very large linear polysaccharides that undergo a complex variety of modifications and are known to play important roles in human development, cell-cell communication and disease. Sequencing of highly sulfated glycosaminoglycan oligosaccharides like heparin and heparan sulfate by liquid chromatography-tandem mass spectrometry (LC-MS/MS) remains challenging because of the presence of multiple isomeric sequences in a complex mixture of oligosaccharides, the difficulties in separation of these isomers, and the facile loss of sulfates in MS/MS. We have previously introduced a method for structural sequencing of heparin/heparan sulfate oligosaccharides involving chemical derivatizations that replace labile sulfates with stable acetyl groups. This chemical derivatization scheme allows the use of reversed phase LC for high-resolution separation and MS/MS for sequencing of isomeric heparan sulfate oligosaccharides. However, because of the large number of analytes present in complex mixtures of heparin/HS oligosaccharides, the resulting LC-MS/MS data sets are large and cannot be annotated with existing glycomics software because of the specifically designed chemical derivatization strategy. We have developed a tool, called GAG-ID, to automate the interpretation of derivatized heparin/heparan sulfate LC-MS/MS data based on a modified multivariate hypergeometric distribution to weight the annotation of more intense peaks. The software is tested on a LC-MS/MS data set collected from a mixture of 21 synthesized heparan sulfate tetrasaccharides. By testing the discrimination of scoring with this system, we show that stratifying peaks into different intensity classes benefits the discrimination of scoring, and GAG-ID is able to properly assign all 21 synthetic tetrasaccharides in a defined mixture from a single LC-MS/MS run.

Carbon Fixation Driven by Molecular Hydrogen Results in Chemolithoautotrophically Enhanced Growth of Helicobacter pylori.

UNLABELLED: A molecular hydrogen (H2)-stimulated, chemolithoautotrophic growth mode for the gastric pathogen Helicobacter pylori is reported. In a culture medium containing peptides and amino acids, H2-supplied cells consistently achieved 40 to 60% greater growth yield in 16 h and accumulated 3-fold more carbon from [(14)C]bicarbonate (on a per cell basis) in a 10-h period than cells without H2 Global proteomic comparisons of cells supplied with different atmospheric conditions revealed that addition of H2 led to increased amounts of hydrogenase and the biotin carboxylase subunit of acetyl coenzyme A (acetyl-CoA) carboxylase (ACC), as well as other proteins involved in various cellular functions, including amino acid metabolism, heme synthesis, or protein degradation. In agreement with this result, H2-supplied cells contained 3-fold more ACC activity than cells without H2 Other possible carbon dioxide (CO2) fixation enzymes were not up-expressed under the H2-containing atmosphere. As the gastric mucus is limited in carbon and energy sources and the bacterium lacks mucinase, this new growth mode may contribute to the persistence of the pathogen in vivo This is the first time that chemolithoautotrophic growth is described for a pathogen. IMPORTANCE: Many pathogens must survive within host areas that are poorly supplied with carbon and energy sources, and the gastric pathogen Helicobacter pylori resides almost exclusively in the nutritionally stringent mucus barrier of its host. Although this bacterium is already known to be highly adaptable to gastric niches, a new aspect of its metabolic flexibility, whereby molecular hydrogen use (energy) is coupled to carbon dioxide fixation (carbon acquisition) via a described carbon fixation enzyme, is shown here. This growth mode, which supplements heterotrophy, is termed chemolithoautotrophy and has not been previously reported for a pathogen.

Proteomic analysis of the acidocalcisome, an organelle conserved from bacteria to human cells.

Acidocalcisomes are acidic organelles present in a diverse range of organisms from bacteria to human cells. In this study acidocalcisomes were purified from the model organism Trypanosoma brucei, and their protein composition was determined by mass spectrometry. The results, along with those that we previously reported, show that acidocalcisomes are rich in pumps and transporters, involved in phosphate and cation homeostasis, and calcium signaling. We validated the acidocalcisome localization of seven new, putative, acidocalcisome proteins (phosphate transporter, vacuolar H+-ATPase subunits a and d, vacuolar iron transporter, zinc transporter, polyamine transporter, and acid phosphatase), confirmed the presence of six previously characterized acidocalcisome proteins, and validated the localization of five novel proteins to different subcellular compartments by expressing them fused to epitope tags in their endogenous loci or by immunofluorescence microscopy with specific antibodies. Knockdown of several newly identified acidocalcisome proteins by RNA interference (RNAi) revealed that they are essential for the survival of the parasites. These results provide a comprehensive insight into the unique composition of acidocalcisomes of T. brucei, an important eukaryotic pathogen, and direct evidence that acidocalcisomes are especially adapted for the accumulation of polyphosphate.

Reliable LC-MS quantitative glycomics using iGlycoMab stable isotope labeled glycans as internal standards.

Glycans have numerous functions in various biological processes and participate in the progress of diseases. Reliable quantitative glycomic profiling techniques could contribute to the understanding of the biological functions of glycans, and lead to the discovery of potential glycan biomarkers for diseases. Although LC-MS is a powerful analytical tool for quantitative glycomics, the variation of ionization efficiency and MS intensity bias are influencing quantitation reliability. Internal standards can be utilized for glycomic quantitation by MS-based methods to reduce variability. In this study, we used stable isotope labeled IgG2b monoclonal antibody, iGlycoMab, as an internal standard to reduce potential for errors and to reduce variabililty due to sample digestion, derivatization, and fluctuation of nanoESI efficiency in the LC-MS analysis of permethylated N-glycans released from model glycoproteins, human blood serum, and breast cancer cell line. We observed an unanticipated degradation of isotope labeled glycans, tracked a source of such degradation, and optimized a sample preparation protocol to minimize degradation of the internal standard glycans. All results indicated the effectiveness of using iGlycoMab to minimize errors originating from sample handling and instruments.

Interlaboratory study on differential analysis of protein glycosylation by mass spectrometry: the ABRF glycoprotein research multi-institutional study 2012.

One of the principal goals of glycoprotein research is to correlate glycan structure and function. Such correlation is necessary in order for one to understand the mechanisms whereby glycoprotein structure elaborates the functions of myriad proteins. The accurate comparison of glycoforms and quantification of glycosites are essential steps in this direction. Mass spectrometry has emerged as a powerful analytical technique in the field of glycoprotein characterization. Its sensitivity, high dynamic range, and mass accuracy provide both quantitative and sequence/structural information. As part of the 2012 ABRF Glycoprotein Research Group study, we explored the use of mass spectrometry and ancillary methodologies to characterize the glycoforms of two sources of human prostate specific antigen (PSA). PSA is used as a tumor marker for prostate cancer, with increasing blood levels used to distinguish between normal and cancer states. The glycans on PSA are believed to be biantennary N-linked, and it has been observed that prostate cancer tissues and cell lines contain more antennae than their benign counterparts. Thus, the ability to quantify differences in glycosylation associated with cancer has the potential to positively impact the use of PSA as a biomarker. We studied standard peptide-based proteomics/glycomics methodologies, including LC-MS/MS for peptide/glycopeptide sequencing and label-free approaches for differential quantification. We performed an interlaboratory study to determine the ability of different laboratories to correctly characterize the differences between glycoforms from two different sources using mass spectrometry methods. We used clustering analysis and ancillary statistical data treatment on the data sets submitted by participating laboratories to obtain a consensus of the glycoforms and abundances. The results demonstrate the relative strengths and weaknesses of top-down glycoproteomics, bottom-up glycoproteomics, and glycomics methods.

Liquid chromatography-selected reaction monitoring (LC-SRM) approach for the separation and quantitation of sialylated N-glycans linkage isomers.

The study of N-linked glycans is among the most challenging bioanalytical tasks because of their complexity and variety. The presence of glycoform families that differ only in branching and/or linkage position makes the identification and quantitation of individual glycans exceedingly difficult. Quantitation of these individual glycans is important because changes in the abundance of these isomers are often associated with significant biomedical events. For instance, previous studies have shown that the ratio of α2-3 to α2-6 linked sialic acid (SA) plays an important role in cancer biology. Consequently, quantitative methods to detect alterations in the ratios of glycans based on their SA linkages could serve as a diagnostic tool in oncology, yet traditional glycomic profiling cannot readily differentiate between these linkage isomers. Here, we present a liquid chromatography-selected reaction monitoring (LC-SRM) approach that we demonstrate is capable of quantitating the individual SA linkage isomers. The LC method is capable of separating sialylated N-glycan isomers differing in α2-3 and α2-6 linkages using a novel superficially porous particle (Fused-Core) Penta-HILIC (hydrophilic interaction liquid chromatography) column. SRM detection provides the relative quantitation of each SA linkage isomer, and minimizes interferences from coeluting glycans that are problematic for UV/Fluorescence based quantitation. With our approach, the relative quantitation of each SA linkage isomer is obtained from a straightforward liquid chromatography-mass spectrometry (LC-MS) experiment.

Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetic homogalacturonan:galacturonosyltransferase complex.

Plant cell wall pectic polysaccharides are arguably the most complex carbohydrates in nature. Progress in understanding pectin synthesis has been slow due to its complex structure and difficulties in purifying and expressing the low-abundance, Golgi membrane-bound pectin biosynthetic enzymes. Arabidopsis galacturonosyltransferase (GAUT) 1 is an α-1,4-galacturonosyltransferase (GalAT) that synthesizes homogalacturonan (HG), the most abundant pectic polysaccharide. We now show that GAUT1 functions in a protein complex with the homologous GAUT7. Surprisingly, although both GAUT1 and GAUT7 are type II membrane proteins with single N-terminal transmembrane-spanning domains, the N-terminal region of GAUT1, including the transmembrane domain, is cleaved in vivo. This raises the question of how the processed GAUT1 is retained in the Golgi, the site of HG biosynthesis. We show that the anchoring of GAUT1 in the Golgi requires association with GAUT7 to form the GAUT1:GAUT7 complex. Proteomics analyses also identified 12 additional proteins that immunoprecipitate with the GAUT1:GAUT7 complex. This study provides conclusive evidence that the GAUT1:GAUT7 complex is the catalytic core of an HG:GalAT complex and that cell wall matrix polysaccharide biosynthesis occurs via protein complexes. The processing of GAUT1 to remove its N-terminal transmembrane domain and its anchoring in the Golgi by association with GAUT7 provides an example of how specific catalytic domains of plant cell wall biosynthetic glycosyltransferases could be assembled into protein complexes to enable the synthesis of the complex and developmentally and environmentally plastic plant cell wall.

Mitochondrial membrane complex that contains proteins necessary for tRNA import in Trypanosoma brucei.

The mitochondrial genome of Trypanosoma brucei does not contain genes encoding tRNAs; instead this protozoan parasite must import nuclear-encoded tRNAs from the cytosol for mitochondrial translation. Previously, it has been shown that mitochondrial tRNA import requires ATP hydrolysis and a proteinaceous mitochondrial membrane component. However, little is known about the mitochondrial membrane proteins involved in tRNA binding and translocation into the mitochondrion. Here we report the purification of a mitochondrial membrane complex using tRNA affinity purification and have identified several protein components of the putative tRNA translocon by mass spectrometry. Using an in vivo tRNA import assay in combination with RNA interference, we have verified that two of these proteins, Tb11.01.4590 and Tb09.v1.0420, are involved in mitochondrial tRNA import. Using Protein C Epitope -Tobacco Etch Virus-Protein A Epitope (PTP)-tagged Tb11.01.4590, additional associated proteins were identified including Tim17 and other mitochondrial proteins necessary for mitochondrial protein import. Results presented here identify and validate two novel protein components of the putative tRNA translocon and provide additional evidence that mitochondrial tRNA and protein import have shared components in trypanosomes.

Catalytic mechanism of Sep-tRNA:Cys-tRNA synthase: sulfur transfer is mediated by disulfide and persulfide.

Sep-tRNA:Cys-tRNA synthase (SepCysS) catalyzes the sulfhydrylation of tRNA-bound O-phosphoserine (Sep) to form cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) in methanogens that lack the canonical cysteinyl-tRNA synthetase (CysRS). A crystal structure of the Archaeoglobus fulgidus SepCysS apoenzyme provides information on the binding of the pyridoxal phosphate cofactor as well as on amino acid residues that may be involved in substrate binding. However, the mechanism of sulfur transfer to form cysteine was not known. Using an in vivo Escherichia coli complementation assay, we showed that all three highly conserved Cys residues in SepCysS (Cys(64), Cys(67), and Cys(272) in the Methanocaldococcus jannaschii enzyme) are essential for the sulfhydrylation reaction in vivo. Biochemical and mass spectrometric analysis demonstrated that Cys(64) and Cys(67) form a disulfide linkage and carry a sulfane sulfur in a portion of the enzyme. These results suggest that a persulfide group (containing a sulfane sulfur) is the proximal sulfur donor for cysteine biosynthesis. The presence of Cys(272) increased the amount of sulfane sulfur in SepCysS by 3-fold, suggesting that this Cys residue facilitates the generation of the persulfide group. Based upon these findings, we propose for SepCysS a sulfur relay mechanism that recruits both disulfide and persulfide intermediates.

Biosynthesis of 4-thiouridine in tRNA in the methanogenic archaeon Methanococcus maripaludis.

4-Thiouridine (s(4)U) is a conserved modified nucleotide at position 8 of bacterial and archaeal tRNAs and plays a role in protecting cells from near-UV killing. Escherichia coli employs the following two enzymes for its synthesis: the cysteine desulfurase IscS, which forms a Cys persulfide enzyme adduct from free Cys; and ThiI, which adenylates U8 and transfers sulfur from IscS to form s(4)U. The C-terminal rhodanese-like domain (RLD) of ThiI is responsible for the sulfurtransferase activity. The mechanism of s(4)U biosynthesis in archaea is not known as many archaea lack cysteine desulfurase and an RLD of the putative ThiI. Using the methanogenic archaeon Methanococcus maripaludis, we show that deletion of ThiI (MMP1354) abolished the biosynthesis of s(4)U but not of thiamine. MMP1354 complements an Escherichia coli ΔthiI mutant for s(4)U formation, indicating that MMP1354 is sufficient for sulfur incorporation into s(4)U. In the absence of an RLD, MMP1354 uses Cys(265) and Cys(268) located in the PP-loop pyrophosphatase domain to generate persulfide and disulfide intermediates for sulfur transfer. In vitro assays suggest that S(2-) is a physiologically relevant sulfur donor for s(4)U formation catalyzed by MMP1354 (K(m) for Na(2)S is ∼1 mm). Thus, methanogenic archaea developed a strategy for sulfur incorporation into s(4)U that differs from bacteria; this may be an adaptation to life in sulfide-rich environments.

Recommendations for mass spectrometry data quality metrics for open access data (corollary to the Amsterdam Principles).

Policies supporting the rapid and open sharing of proteomic data are being implemented by the leading journals in the field. The proteomics community is taking steps to ensure that data are made publicly accessible and are of high quality, a challenging task that requires the development and deployment of methods for measuring and documenting data quality metrics. On September 18, 2010, the United States National Cancer Institute convened the "International Workshop on Proteomic Data Quality Metrics" in Sydney, Australia, to identify and address issues facing the development and use of such methods for open access proteomics data. The stakeholders at the workshop enumerated the key principles underlying a framework for data quality assessment in mass spectrometry data that will meet the needs of the research community, journals, funding agencies, and data repositories. Attendees discussed and agreed up on two primary needs for the wide use of quality metrics: 1) an evolving list of comprehensive quality metrics and 2) standards accompanied by software analytics. Attendees stressed the importance of increased education and training programs to promote reliable protocols in proteomics. This workshop report explores the historic precedents, key discussions, and necessary next steps to enhance the quality of open access data. By agreement, this article is published simultaneously in the Journal of Proteome Research, Molecular and Cellular Proteomics, Proteomics, and Proteomics Clinical Applications as a public service to the research community. The peer review process was a coordinated effort conducted by a panel of referees selected by the journals.

An integrated transcriptomic and computational analysis for biomarker identification in gastric cancer.

This report describes an integrated study on identification of potential markers for gastric cancer in patients' cancer tissues and sera based on: (i) genome-scale transcriptomic analyses of 80 paired gastric cancer/reference tissues and (ii) computational prediction of blood-secretory proteins supported by experimental validation. Our findings show that: (i) 715 and 150 genes exhibit significantly differential expressions in all cancers and early-stage cancers versus reference tissues, respectively; and a substantial percentage of the alteration is found to be influenced by age and/or by gender; (ii) 21 co-expressed gene clusters have been identified, some of which are specific to certain subtypes or stages of the cancer; (iii) the top-ranked gene signatures give better than 94% classification accuracy between cancer and the reference tissues, some of which are gender-specific; and (iv) 136 of the differentially expressed genes were predicted to have their proteins secreted into blood, 81 of which were detected experimentally in the sera of 13 validation samples and 29 found to have differential abundances in the sera of cancer patients versus controls. Overall, the novel information obtained in this study has led to identification of promising diagnostic markers for gastric cancer and can benefit further analyses of the key (early) abnormalities during its development.

Embracing Innovation and Accessibility in Biomolecular Research Publishing.

We are thrilled to share the latest developments at the Journal of Biomolecular Techniques (JBT), your esteemed peer-reviewed publication dedicated to advancing biotechnology research. Since its inception, JBT has been committed to promoting the pivotal role that biotechnology plays in contemporary scientific endeavors, fostering knowledge exchange among biomolecular resource facilities, and communicating the groundbreaking research conducted by the Association's Research Groups, members, and other investigators.

Do I Need to Trypsin Digest Before Releasing IgG Glycans With PNGase-F?

Immunoglobulin G (IgG) is the main immunoglobulin in human serum, and its biological activity is modulated by glycosylation on its fragment crystallizable region. Glycosylation of IgGs has shown to be related to aging, disease progression, protein stability, and many other vital processes. A common approach to analyze IgG glycosylation involves the release of the N-glycans by PNGase F, which cleaves the linkage between the asparagine residue and the innermost N-acetylglucosamine (GlcNAc) of all N-glycans except those containing a 3-linked fucose attached to the core GlcNAc. The biological significance of these glycans necessitates the development of accurate methods for their characterization and quantification. Currently, researchers either perform PNGase F deglycosylation on intact or trypsin-digested IgGs. Those who perform PNGase F deglycosylation on trypsin-digested IgGs argue that proteolysis is needed to reduce steric hindrance, whereas the other group states that this step is not needed, and the proteolytic step only adds time. There is minimal experimental evidence supporting either assumption. The importance of obtaining complete glycan release for accurate quantitation led us to investigate the kinetics of this deglycosylation reaction for intact IgGs and IgG glycopeptides. Statistically significant differences in the rate of deglycosylation performed on intact IgGs and trypsin-digested IgGs were determined, and the rate of PNGase F deglycosylation on trypsin-digested IgGs was found to be 3- to 4-times faster than on intact IgG.

A Survey on Core Flow Cytometry Facilities: Instrument Maintenance, Usage, and Funding.

Flow cytometry is a powerful tool that finds applications in various fields such as immunology, molecular biology, cancer biology, virology, and infectious disease monitoring. A significant portion of the research in these disciplines is supported by flow cytometry shared resource laboratories (SRLs). There are several types of flow cytometers available for use in SRLs, including analyzers, sorters, imaging flow cytometers, and mass cytometers. Each type has different challenges when it comes to maintenance and life expectancy. An independent online survey was conducted to better understand instrument maintenance and turnover in flow cytometry SRLs. Questions regarding instrument uptime (availability), its usage, routine maintenance, and cost associated with it were addressed. The respondents also answered questions pertaining to the frequency of deep cleaning of the instrument and quality control. In addition, the survey queried about the source of funding used to purchase the instruments and possible reasons for a replacement. Presented herein are the results compiled from 146 core facilities that provide a look at the operation within a typical SRL, with the responses reflecting researchers' experiences with handling flow cytometers.

Recommendations for mass spectrometry data quality metrics for open access data (corollary to the Amsterdam principles).

Policies supporting the rapid and open sharing of proteomic data are being implemented by the leading journals in the field. The proteomics community is taking steps to ensure that data are made publicly accessible and are of high quality, a challenging task that requires the development and deployment of methods for measuring and documenting data quality metrics. On September 18, 2010, the U.S. National Cancer Institute (NCI) convened the "International Workshop on Proteomic Data Quality Metrics" in Sydney, Australia, to identify and address issues facing the development and use of such methods for open access proteomics data. The stakeholders at the workshop enumerated the key principles underlying a framework for data quality assessment in mass spectrometry data that will meet the needs of the research community, journals, funding agencies, and data repositories. Attendees discussed and agreed upon two primary needs for the wide use of quality metrics: (i) an evolving list of comprehensive quality metrics and (ii) standards accompanied by software analytics. Attendees stressed the importance of increased education and training programs to promote reliable protocols in proteomics. This workshop report explores the historic precedents, key discussions, and necessary next steps to enhance the quality of open access data. By agreement, this article is published simultaneously in Proteomics, Proteomics Clinical Applications, Journal of Proteome Research, and Molecular and Cellular Proteomics, as a public service to the research community. The peer review process was a coordinated effort conducted by a panel of referees selected by the journals.

Proteome analysis of Cry4Ba toxin-interacting Aedes aegypti lipid rafts using geLC-MS/MS.

Lipid rafts are microdomains in the plasma membrane of eukaryotic cells. Among their many functions, lipid rafts are involved in cell toxicity caused by pore forming bacterial toxins including Bacillus thuringiensis (Bt) Cry toxins. We isolated lipid rafts from brush border membrane vesicles (BBMV) of Aedes aegypti larvae as a detergent resistant membrane (DRM) fraction on density gradients. Cholesterol, aminopeptidase (APN), alkaline phosphatase (ALP) and the raft marker flotillin were preferentially partitioned into the lipid raft fraction. When mosquitocidal Cry4Ba toxin was preincubated with BBMV, Cry4Ba localized to lipid rafts. A proteomic approach based on one-dimensional gel electrophoresis, in-gel trypsin digestion, followed by liquid chromatography-mass spectrometry (geLC-MS/MS) identified a total of 386 proteins. Of which many are typical lipid raft marker proteins including flotillins and glycosylphosphatidylinositol (GPI)-anchored proteins. Identified raft proteins were annotated in silico for functional and physicochemical characteristics. Parameters such as distribution of isoelectric point, molecular mass, and predicted post-translational modifications relevant to lipid raft proteins (GPI anchorage and myristoylation or palmitoylation) were analyzed for identified proteins in the DRM fraction. From a functional point of view, this study identified proteins implicated in Cry toxin interactions as well as membrane-associated proteins expressed in the mosquito midgut that have potential relevance to mosquito biology and vector management.

Proteomic analysis of ripening tomato fruit infected by Botrytis cinerea.

Botrytis cinerea, a model necrotrophic fungal pathogen that causes gray mold as it infects different organs on more than 200 plant species, is a significant contributor to postharvest rot in fresh fruit and vegetables, including tomatoes. By describing host and pathogen proteomes simultaneously in infected tissues, the plant proteins that provide resistance and allow susceptibility and the pathogen proteins that promote colonization and facilitate quiescence can be identified. This study characterizes fruit and fungal proteins solubilized in the B. cinerea-tomato interaction using shotgun proteomics. Mature green, red ripe wild type and ripening inhibited (rin) mutant tomato fruit were infected with B. cinerea B05.10, and the fruit and fungal proteomes were identified concurrently 3 days postinfection. One hundred eighty-six tomato proteins were identified in common among red ripe and red ripe-equivalent ripening inhibited (rin) mutant tomato fruit infected by B. cinerea. However, the limited infections by B. cinerea of mature green wild type fruit resulted in 25 and 33% fewer defense-related tomato proteins than in red and rin fruit, respectively. In contrast, the ripening stage of genotype of the fruit infected did not affect the secreted proteomes of B. cinerea. The composition of the collected proteins populations and the putative functions of the identified proteins argue for their role in plant-pathogen interactions.