Enzyme-catalyzed 18O2-labeling offers a universal strategy for uniform labeling of all peptides from any kind of proteins, including post-translationally modified proteins. It is applicable to clinical samples with unrivaled sensitivity. This review discusses strengths and limitations, and advocates the separation of proteolysis from the labeling step. Continued advances in software will facilitate widespread use of enzyme-catalyzed 18O2-labeling to determine changes in protein abundances.Keywords: 18O-labeling; absolute quantitation; affinity fractionation; biofilms; clinical proteomics; formalin-fixed paraffin; glycoproteins; isotope labeling; laser capture micro-dissection; mitochondria; plasma membrane; plasma proteome; protein cross-linking; quantitative proteomics; tissue proteomics;

In this report, we use a proteomic strategy to identify glycoproteins on the surface of exosomes derived from myeloid-derived suppressor cells (MDSCs), and then test if selected glycoproteins contribute to exosome-mediated chemotaxis and migration of MDSCs. We report successful modification of a surface chemistry method for use with exosomes and identify 21 surface N-glycoproteins on exosomes released by mouse mammary carcinoma-induced MDSCs. These glycoprotein identities and functionalities are compared with 93 N-linked glycoproteins identified on the surface of the parental cells. As with the lysate proteomes examined previously, the exosome surface N-glycoproteins are primarily a subset of the glycoproteins on the surface of the suppressor cells that released them, with related functions and related potential as therapeutic targets. The “don’t eat me” molecule CD47 and its binding partners thrombospondin-1 (TSP1) and signal regulatory protein α (SIRPα) were among the surface N-glycoproteins detected. Functional bioassays using antibodies to these three molecules demonstrated that CD47, TSP1, and to a lesser extent SIRPα facilitate exosome-mediated MDSC chemotaxis and migration.Keywords: cell surface capture; exosomes; myeloid derived suppressor cells; N-glycoproteome;

Unexpected tryptic cleavage has been characterized at modified K48 residues in polyubiquitins. In particular, the tryptic products of all seven of the lysine-linked dimers of ubiquitin and of three trimers—linear Ub–48Ub–48Ub, linear Ub–63Ub–63Ub, and the branched trimer [Ub]2–6,48Ub—have been analyzed. In addition to the peptide products expected under commonly used tryptic conditions, we observe that peptides are formed with an unexpected ε-glycinylglycinyl-Lys carboxyl terminus when the site of linkage is Lys48. Trypsin from three different commercial sources exhibited this aberration. Initial cleavage at R74 is proposed in a distal ubiquitin to produce a glycinylglycinyl-lysine residue which is bound by trypsin.

•A workflow has been developed for successful top–down analysis of low mass proteins using HPLC-MS/MS.•Multiple proteoforms are identified of the pro-inflammatory mediators S100 A8 and A9 in exosomes shed by MDSC cells that suppress the immune response to cancer.•Histone proteoforms are found to constitute 56% of the protein cargo carried by these exosomes.•Four histone variants are shown to carry a rare processing modification.Top–down analysis is reported for a portion of the protein cargo of exosomes shed by myeloid-derived suppressor cells that participate in intracellular signaling in the tumor microenvironment. Instrument mass resolution limited the study to proteins of molecular masses below 30 kDa. A two-step fractionation strategy was used, including open tubular gel electrophoresis and C3 reverse phase high performance liquid chromatography. Twenty-one unique proteins were identified among more than 200 proteoforms, and comprising primarily two functionally important protein families: the S100 proinflammatory mediators and an abundance of histones. Fifty-six percent of the total protein in these exosomes was determined to comprise histones, of which H2B variants contribute 42%.

Myeloid-derived suppressor cells (MDSC) are present in most cancer patients where they inhibit natural anti-tumor immunity and are an obstacle to anti-cancer immunotherapies. They mediate immune suppression through their production of proteins and soluble mediators that prevent the activation of tumor-reactive T lymphyocytes, polarize macrophages toward a tumor-promoting phenotype, and facilitate angiogenesis. The accumulation and suppressive potency of MDSC is regulated by inflammation within the tumor microenvironment. Recently exosomes have been proposed to act as intercellular communicators, carrying active proteins and other molecules between sender cells and receiver cells. In this report we describe the proteome of exosomes shed by MDSC induced in BALB/c mice by the 4T1 mammary carcinoma. Using bottom-up proteomics, we have identified 412 proteins. Spectral counting identified 63 proteins whose abundance was altered >2-fold in the inflammatory environment. The pro-inflammatory proteins S100A8 and S100A9, previously shown to be secreted by MDSC and to be chemotactic for MDSC, are abundant in MDSC-derived exosomes. Bioassays reveal that MDSC-derived exosomes polarize macrophages toward a tumor-promoting type 2 phenotype, in addition to possessing S100A8/A9 chemotactic activity. These results suggest that some of the tumor-promoting functions of MDSC are implemented by MDSC-shed exosomes.

Proteomic and other characterization of plasma membrane proteins is made difficult by their low abundance, hydrophobicity, frequent carboxylation, and dynamic population. We and others have proposed that underrepresentation in LC-MS/MS analysis can be partially compensated by enriching the plasma membrane and its proteins using cationic nanoparticle pellicles. The nanoparticles increase the density of plasma membrane sheets and thus enhance separation by centrifugation from other lysed cellular components. Herein, we test the hypothesis that the use of nanoparticles with increased densities can provide enhanced enrichment of plasma membrane proteins for proteomic analysis. Multiple myeloma cells were grown and coated in suspension with three different pellicles of three different densities and both pellicle coated and uncoated suspensions analyzed by high-throughput LC-MS/MS. Enrichment was evaluated by the total number and the spectral counts of identified plasma membrane proteins.

This study is reported on the effect of nanowire density on the ease of pellicle formation and the enrichment of plasma membrane (PM) proteins for analysis by mass spectrometry. An optimized synthesis is reported for iron silicate nanowires (ISNW) with a narrow size range of 900 ± 400 nm in length and 200 nm diameter. The nanowires were coated with Al2O3 and used to form pellicles around suspended multiple myeloma cells, which acted as a model for cells recovered from tissue samples. Lighter alumina-coated silica nanowires were also synthesized (Kim et al., doi:10.2217/NNM.13.96, 2013), which allowed a comparison of the construction of the two pellicles and of the effect of nanowire density on PM enrichment. Evidence is offered that the dense nanowire pellicle does not crush or distort these mammalian cells. Finally, the pellicles were incorporated into a mass spectrometry-based proteomic workflow to analyze transmembrane proteins in the PM. In contrast to a prior comparison of the effect of density with nanoparticles pellicles (Choksawangkarn et al. J Proteome Res 455 12:1134–1141, 2013), nanowire density was not found to significantly affect the enrichment of the PM. However, nanowires with a favorable aspect for pellicle formation are more easily and reliably produced with iron silicate than with silica. In addition, the method for pellicle formation was optimized through the use of ISNW, which is crucial to the improvement of PM protein enrichment and analysis.

Strategies for the rapid and reliable analysis of microorganisms have been sought to meet national needs in defense, homeland security, space exploration, food and water safety, and clinical diagnosis. Mass spectrometry has long been a candidate technique because it is extremely rapid and can provide highly specific information. It has excellent sensitivity. Molecular and fragment ion masses provide detailed fingerprints, which can also be interpreted. Mass spectrometry is also a broad band method—everything has a mass—and it is automatable. Mass spectrometry is a physiochemical method that is orthogonal and complementary to biochemical and morphological methods used to characterize microorganisms.

Proteomic studies of plasma membrane proteins are challenged by the limited solubility of these proteins and the limited activity of proteolytic enzymes in solubilizing agents such as SDS. In this work, we have evaluated three bottom-up workflows to obtain tryptic peptides from plasma membrane proteins solubilized with 2% SDS. The workflows are in-gel digestion, in-solution digestion, and on-filter digestion. The efficiencies of these strategies, optimized to employ different matrices for trypsin cleavage, were compared using a plasma membrane sample enriched from multiple myeloma cells using a nanoparticle pellicle. On the basis of the number of proteins identified, number of transmembrane proteins identified, hydrophobicity, and spectral count per protein, the workflow that uses in-gel digestion is the most advantageous approach for analysis of plasma membrane proteins.

In addition to its critical role in normal cell function, growth, and metabolism, zinc is implicated as a major factor in the development and progression of many pathological conditions and diseases. Despite this importance of zinc, many important factors, processes, and mechanisms of the physiology, biochemistry, and molecular biology of zinc remain unknown. Especially important is the unresolved issue regarding the mechanism and process of the trafficking, transport, and reactivity of zinc in cells; especially in mammalian cells. This presentation focuses on the concept that, due to the existence of a negligible pool of free Zn2+ ions in the mammalian cell environment, the trafficking, transport and reactivity of zinc occurs via a direct exchange of zinc from donor Zn-ligands to acceptor ligands. This Zn exchange process occurs without the requirement for production of free Zn2+ ions. The direct evidence from mammalian cell studies is presented in support of the operation of the direct Zn-ligand exchange mechanism. The paper also provides important information and conditions that should be considered and employed in the conduct of studies regarding the role and effects of zinc in biological/biomedical research; and in its clinical interpretation and application.We show that the transport, trafficking, and reactivity of zinc occur via a direct Zn ligand exchange process.

Microwave assisted acid cleavage was applied directly to intact adenovirus type 5 to achieve denaturation and proteolysis in a single reaction. The speed of the digestion, coupled with the simplicity of MALDI analysis, allowed peptide products to be profiled in less than 5 min. Identification of peptides from a range of proteins by MALDI-TOF confirms that both denaturation and proteolysis were achieved using low concentrations of acetic acid (12.5%) and short incubations (1.5–2 min) at high temperatures (140 °C). These conditions favor production of peptides that carry Asp on their C-termini. When this cleavage reaction is carried out in highly enriched H218O, a single atom of 18O is introduced site-specifically into the carboxyl terminal. The labeling reaction is applied to label reporter peptides from human adenovirus type 5 harvested from HeLa cells. Small peptide products of endogenous processing were also observed.

This report demonstrates the application of a capillary LC-LTQ-orbitrap system to provide automated middle-down analysis of proteolytic peptides in the mass range 3000 to 10 000 Da. The novel workflow combines an underutilized method in the orbitrap—high resolution, mass-accurate product ion measurements—with software tailored to search such data (ProSightPC 2.0) and an Asp-selective chemical cleavage approach that generates peptides across an extended mass range. The strategy using high resolution mass measurements on both precursor and product ions is analogous to that widely used on FT-ICR analyzers. The approach is demonstrated in an analysis of the highly basic ribosomal proteome isolated from human MCF7 cancer cells.

MALDI mass spectrometry-based systems for rapid characterization of microorganisms in biodefense or medical diagnostics usually detect intact proteins in the 5000−20,000 Da range. To evaluate the reliability of species discrimination, and also for forensic applications, it is important that these biomarker proteins be identified. In the present study we apply high resolution tandem mass analysis on an Orbitrap and top-down bioinformatics to identify major biomarker proteins observed in MALDI spectra of intact bacteria for which little genomic or protein sequence information is available. The strategy depends on recognition of proteins with very high homology in related (sequenced) species, making it possible to place unsequenced organisms in their correct phylogenetic context. We show that this rapid proteomics based approach to phylogenetic characterization produces similar results to the traditional techniques, and may even be applied to target organisms of undetermined taxonomy. We further discuss important issues in combining genomics/proteomics databases and MALDI MS for the rapid characterization of microorganisms.

Microwave-accelerated proteolysis using acetic acid has been shown to occur specifically on either or both sides of aspartic acid residues. This chemical cleavage has been applied to ovalbumin and several model peptides to test the effect on some of the more common post-translational modifications. No oxidation of methionine or cysteine was observed; however, hydrolysis of phosphate groups proceeds at a detectable rate. Acid cleavage was also extended to the yeast ribosome model proteome, where it provided information on 74% of that proteome. Aspartic acid occurs across the proteome with approximately half the frequency of the combined occurrence of the trypsin residues lysine and arginine, and implications of this are considered.

This laboratory has introduced a chemical method for residue-specific protein cleavage and has provided a preliminary assessment of the suitability of microwave-accelerated acid cleavage as a proteomic tool. This report is a continuing assessment of the fate of common protein modifications in microwave-accelerated acid cleavage. We have examined the cleavage of ribonuclease A and the related N-linked glycoprotein ribonuclease B, and the O-linked glycoprotein alpha crystallin A chain, using MALDI-TOF and LC–ESI-MS to identify the peptide products. RNase A and B each contains four disulfide bonds, and the addition of a reducing reagent, such as dithiothreitol, was found to be required to achieve efficient acidic proteolysis. The linkage of the glycosidic group to the asparagine side chain in ribonuclease B was found not to be cleaved by brief microwave treatment in 12.5% acetic acid. The distribution of the heterogeneous carbohydrate side chain in the glycopeptide products of acid cleavage was compared to that of the glycopeptide products of tryptic digestion. Hydrolysis within the carbohydrate chain itself is minimal under the conditions used. The O-linked side chain on alpha crystalline A was found to be cleaved during acid cleavage of the protein.

Forward and reverse 18O labeling are integrated with solution isoelectric focusing and capillary LC-tandem mass spectrometry to evaluate a new strategy for quantitative proteomics and to study abundance changes in mitochondrial proteins associated with drug resistance in MCF-7 human cancer cells. Galectin-3 binding protein, which is involved in apoptosis, was detected only in the resistant cell line, as a result of reverse labeling. Among 278 proteins identified, 12 were detected with abundances altered at least 2-fold.