Download the Free Unbound MEDLINE PubMed App to your smartphone or tablet.
Available for iPhone, iPad, iPod touch, and Android.
Molecular cellular proteomics [journal]
- In Vivo Assessment of Protease Dynamics in Cutaneous Wound Healing by Degradomics Analysis of Porcine Wound Exudates. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Dec 16.
Proteases control complex tissue responses by modulating inflammation, cell proliferation and migration, and matrix remodeling. All these processes are orchestrated in cutaneous wound healing to restore the skin's barrier function upon injury. Altered protease activity has been implicated in the pathogenesis of healing impairments, and proteases are important targets in diagnosis and therapy of this pathology. Global assessment of proteolysis at critical turning points after injury will define crucial events in acute healing that might be disturbed in healing disorders. As optimal biospecimens, wound exudates contain an ideal proteome to detect extracellular proteolytic events, are non-invasively accessible, and can be collected at multiple time points along the healing process from the same wound in the clinics. In this study, we applied multiplexed Terminal Amine Isotopic Labeling of Substrates (TAILS) to globally assess proteolysis in early phases of cutaneous wound healing. By quantitative analysis of proteins and protein N termini in wound fluids from a clinically relevant pig wound model, we identified more than 650 proteins and discerned major healing phases through distinctive abundance clustering of markers of inflammation, granulation tissue formation, and re-epithelialization. TAILS revealed a high degree of proteolysis at all time points after injury by detecting almost 1300 N-terminal peptides in ~450 proteins. Quantitative positional proteomics mapped pivotal interdependent processing events in the blood coagulation and complement cascades, temporally discerned clotting and fibrinolysis during the healing process, and detected processing of complement C3 at distinct time points after wounding and by different proteases. Exploiting data on primary cleavage specificities, we related candidate proteases to cleavage events and revealed processing of the integrin adapter protein kindlin-3 by caspase-3, generating new hypotheses for protease-substrate relations in the healing skin wound in vivo. The data have been deposited to the ProteomeXchange Consortium with identifier PXD001198.
- Anti-peptide monoclonal antibodies generated for immuno-MRM assays have a high probability of supporting Western blot and ELISA. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Dec 15.
Immunoaffinity enrichment of peptides coupled to targeted, multiple reaction monitoring-mass spectrometry (immuno-MRM) has recently been developed for quantitative analysis of peptide and protein expression. As part of this technology, antibodies are generated to short, linear, tryptic peptides that are well-suited for detection by mass spectrometry. Despite its favorable analytical performance, a major obstacle to widespread adoption of immuno-MRM is a lack of validated affinity reagents, since commercial antibody suppliers are reluctant to commit resources to producing anti-peptide antibodies for immuno-MRM while the market is much larger for conventional technologies, especially Western blotting and ELISA. Part of this reluctance has been the concern that affinity reagents generated to short, linear, tryptic peptide sequences may not perform well in traditional assays that detect full-length proteins. In this study, we test the feasibility and success rates of generating immuno-MRM monoclonal antibodies (mAbs) (targeting tryptic peptide antigens) that are also compatible with conventional, protein-based immuno-affinity technologies. We generated 40 novel, peptide immuno-MRM assays and determined that the cross-over success rates for using immuno-MRM monoclonals for Western blotting is 58% and for ELISA is 43%, which compare favorably to cross-over success rates amongst conventional immunoassay technologies. These success rates could most likely be increased if conventional and immuno-MRM antigen design strategies were combined, and we suggest a workflow for such a comprehensive approach. Additionally, the 40 novel immuno-MRM assays underwent fit-for-purpose analytical validation, and all mAbs and assays have been made available as a resource to the community via the Clinical Proteomic Tumor Analysis Consortium's (CPTAC) Antibody (http://antibodies.cancer.gov) and Assay Portals (http://assays.cancer.gov), respectively. This study also represents the first determination of the success rate (92%) for generating mAbs for immuno-MRM using a recombinant B cell cloning approach, which is considerably faster than the traditional hybridoma approach.
- Systematically Ranking the Tightness of Membrane Association for Peripheral Membrane Proteins. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Dec 13.
Large scale quantitative evaluation of the tightness of membrane association for non-transmembrane proteins is important for identifying true peripheral membrane proteins with functional significance. Herein, we simultaneously ranked more than 1,000 proteins of the photosynthetic model organism Synechocystis sp. PCC 6803 for their relative tightness of membrane association using a proteomic approach. Using multiple precisely ranked and experimentally verified peripheral subunits of photosynthetic protein complexes as the landmarks, we found that proteins involved in two-component signal transduction systems and transporters are overall tightly associated with the membranes, whereas the associations of ribosomal proteins are much weaker. Moreover, we found that hypothetical proteins containing the same domains generally have similar tightness. This work provided a global view of the structural organization of the membrane proteome with respect to divergent functions, and built the foundation for future investigation of the dynamic membrane proteome reorganization in response to different environmental or internal stimuli.
- A Chemical Proteomics Approach for Global Analysis of Lysine Mono-Methylation. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Dec 11.
Methylation of lysine residues on histone proteins is known to play an important role in chromatin structure and function. However, non-histone protein substrates of this modificationremain largely unknown. An effective approach for system-wide analysis of protein lysine methylation, particularly lysine mono-methylation, is lacking. Here we describe a chemical proteomics approach for global screening for mono-methyllysine substrates, involving chemical propionylation of mono-methylated lysine, affinity enrichment of the modified mono-methylated peptides, and HPLC/MS/MS analysis. Usingthis approach, we identified with high confidence 446 lysine mono-methylation sites in 398proteins, including 3previously unknown histone mono-methylation marks, representing the largest dataset of protein lysine mono-methylationdescribed to date. Our datanot only confirmed previously discovered lysine methylation substrates in the nucleus and spliceosome, but also revealed new substrates associated with diverse biological processes. Thismethod hence offersa powerful approach for dynamic studyof protein lysine mono-methylation under diversecellular conditions and in human diseases.
- Quantitative Nuclear Proteomics Identifies that miR-137-mediated EZH2 Reduction Regulates Resveratrol-induced Apoptosis of Neuroblastoma Cells. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Dec 11.
Neuroblastoma is the most common pediatric extracranial solid tumor with a broad spectrum of clinical behavior and poor prognosis. Despite intensive multimodal therapy, ongoing clinical trials and basic science investigations, neuroblastoma remains a complex medical challenge with a long-term survival rate less than 40%. In our study, we found that resveratrol (3, 5, 4'-trihydroxystilbene, RSV), a naturally occurring phytoalexin, possesses an anticancer activity through blocking cell growth and inducing apoptosis in neuroblastoma cell line Neuro-2a (N-2a) cells. Using stable isotope labeling with amino acids in cell culture (SILAC) and quantitative proteomic analysis, we found that 395 proteins were up-regulated and 302 proteins were down-regulated in the nucleus of N-2a cells treated with RSV. Among these, the polycomb protein histone methyltransferase EZH2 was reduced significantly, which is aberrantly overexpressed in neuroblastoma and crucial to maintain the malignant phenotype of neuroblastoma by epigenetic repression of multiple tumor suppressor genes. EZH2 reduction further led to decreased H3K27me3 level and reactivation of neuroblastoma tumor suppressor genes CLU and NGFR. Disruption EZH2 expression by RNA interference-mediated knockdown or pharmacologic inhibition with DZNep triggered cellular apoptosis in N-2a cells. We found that the up-regulation of miR-137 level was responsible for reduced EZH2 level in tumor suppression induced by RSV. Inhibition of miR-137 expression rescued the cellular apoptosis phenotypes, EZH2 reduction and CLU and NGFR reactivation, associated with RSV treatment. Taken together, our findings present for the first time, an epigenetic mechanism involving miR-137-mediated EZH2 repression in RSV-induced apoptosis and tumor suppression of neuroblastoma, which would provide a key potential therapeutic target in neuroblastoma treatment.
- mzDB: a file format using multiple indexing strategies for the efficient analysis of large LC-MS/MS and SWATH-MS datasets. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Dec 11.
The analysis and management of MS data, especially those generated by data independent MS acquisition, exemplified by SWATH-MS, pose significant challenges for proteomics bioinformatics. The large size and vast amount of information inherent to these datasets need to be properly structured to enable an efficient and straightforward extraction of the signals used to identify specific target peptides. Standard XML based formats are not well suited to large MS data files, e.g., those generated by SWATH-MS, and compromise high-throughput data processing and storing. We developed mzDB, an efficient file format for large MS data sets. It relies on the SQLite software library and consists of a standardized and portable server-less single-file database. An optimized 3D indexing approach is adopted, where the LC-MS coordinates (retention time and m/z), along with the precursor m/z for SWATH-MS data, are used to query the database for data extraction. In comparison with XML formats, mzDB saves ~25% of storage space and improves access times by a factor of 2 fold up to even 2000 fold, depending on the particular data access. Similarly, mzDB shows also slightly to significantly lower access times in comparison with other formats like mz5. Both C++ and Java implementations, converting raw or XML formats to mzDB and providing access methods, will be released under permissive license. mzDB can be easily accessed by the SQLite C library and its drivers for all major languages, and browsed with existing dedicated GUIs. The mzDB described here can boost existing mass spectrometry data analysis pipelines, offering unprecedented performance in terms of efficiency, portability, compactness, and flexibility.
- A proteomic analysis reveals that Snail regulates the expression of the nuclear orphan receptor Nr2f6 and IL-17 to inhibit adipocyte differentiation. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Dec 10.
Adipogenesis requires a differentiation program driven by multiple transcription factors, where PPARγ and C/EBPα play a central role. Recent findings indicate that Snail inhibits adipocyte differentiation in 3T3-L1 and murine mesenchymal stem cells (mMSC). An in-depth quantitative SILAC analysis of the nuclear fraction of Snail-induced alterations of 3T3-L1 cells was carried out. In total, 2251 overlapping proteins were simultaneously quantified in forward and reverse experiments. We observed 574 proteins deregulated by Snail1 using a fold-change ≥1.5, with 111 up- and 463 down-regulated proteins, respectively. Among other proteins, multiple transcription factors such as Trip4, OsmR, Nr2f6, Cbx6 and Prrx1 were down-regulated. Results were validated in 3T3-L1 cells and mMSC cells by western blot and quantitative PCR. Knock-down experiments in 3T3-L1 cells demonstrated that only Nr2f6 (and Trip4 at minor extent) was required for adipocyte differentiation. Ectopic expression of Nr2f6 reversed the effects of Snail1, promoted adipogenesis and inhibited the expression of IL-17. To prove that correlation, we observed that IL-17 and TNFα were among the most up-regulated pro-inflammatory cytokines in Snail-transfected 3T3-L1 and mMSC cells. Furthermore, the blocking of IL-17 activity in Snail-transfected cells promoted adipocyte differentiation, reverting Snail inhibition. In summary, Snail inhibits adipogenesis through a down-regulation of Nr2f6, which in turn facilitates the expression of IL-17, an anti-adipogenic cytokine. These results would support a novel and important role for Snail and Nr2f6 in obesity control.
- Reverse-Polynomial Dilution Calibration Methodology extends Lower Limit of Quantification and Reduces Relative Residual Error in Targeted Peptide Measurements in Blood Plasma. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Dec 9.
Matrix effect is the alteration of an analytes concentration-signal response due to co-existing ion components. With Electrospray Ionization (ESI), matrix effects are believed to be a function of the relative concentrations, ionization efficiency, and solvation energies of the analytes within the ESI droplet. For biological matrices such as plasma, the interactions between droplet components is immensely complex and the effect on analyte signal response not well elucidated. This study comprised of three sequential quantitative analyses: whether there is a generalizable correlation between the range of unique ions in a sample matrix (complexity); the amount of matrix components (concentration); and matrix effect, by comparing an E.coli digest matrix (~2600 protein proteome) with phospholipid depleted human blood plasma, and unfractionated, non-depleted human plasma matrices (~107 proteome) for six human plasma peptide Multiple Reaction Monitoring (MRM) assays. Our dataset demonstrated analyte-specific interactions with matrix complexity and concentration properties resulting in significant ion suppression for all peptides (p<0.01), with non-uniform effects on the ion signals of the analytes and their stable-isotope analogs. These matrix effects were then assessed for translation into relative residual error and precision affects in a low concentration (~0-250ng/mL) range across no-matrix, complex matrix, and highly complex matrix, when a standard addition Stable Isotope Dilution (SID) calibration method was used. Relative residual error (%) and precision (CV%) by SID were within <20%, however error in phospholipid-depleted and non-depleted plasma matrices were significantly higher compared to no-matrix and E.coli matrices (p=0.007). Finally a novel Reverse-Polynomial Dilution (RPD) calibration method with and without phospholipid-depletion was compared to SID for relative residual error and precision. RPD techniques extend the Lower Limit of Quantification and reduce error (p=0.005) in low-concentration plasma peptide assays and is broadly applicable for verification phase Tier 2 multiplexed MRM assay development within the FDA-National Cancer Institute (NCI) biomarker development pipeline.
- Dodecyl maltopyranoside enabled purification of active human GABA type A receptors for deep and direct proteomic sequencing. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Dec 3.
The challenge in high-quality membrane proteomics is all about sample preparation prior to HPLC, and the cell-to-protein step poses a long-standing bottleneck. Traditional protein extraction methods apply ionic or poly-disperse detergents, harsh denaturation, and repeated protein/peptide precipitation/resolubilization afterwards, but suffer low yield, low reproducibility and low sequence coverage. Contrary to attempts to subdue, we resolved this challenge by providing proteins nature-and-activity-promoting conditions throughout preparation. Using 285-kDa hetero-pentameric human GABA type A receptor overexpressed in HEK293 as a model, we describe a n-dodecyl-beta-D-maltopyranoside/cholesteryl hemisuccinate (DDM/CHS)-based affinity purification method, that produced active receptors, supported protease activity, and allowed high performance with both in-gel and direct gel-free proteomic analyses, without detergent removal. Unlike conventional belief that detergents must be removed before HPLC MS, the high-purity low-dose nonionic detergent DDM did not interfere with peptides, and obviated removal or desalting. Sonication or drop-wise addition of detergent robustly solubilized over 90% of membrane pellets. The purification conditions were comparable to those applied in successful crystallizations of most membrane proteins. These results enabled streamlined proteomics of human synaptic membrane proteins, and more importantly, allowed directly coupling proteomics with crystallography to characterize both static and dynamic structures of membrane proteins in crystallization pipelines.
- Abundance-based classifier for the prediction of mass spectrometric peptide detectability upon enrichment. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Dec 3.
The function of a large percentage of proteins is modulated by post-translational modifications (PTMs). Currently, mass spectrometry (MS) is the only proteome-wide technology that can identify PTMs. Unfortunately, the inability to detect a PTM by MS is not proof that the modification is not present. The detectability of peptides varies significantly making MS potentially blind to a large fraction of peptides. Learning from published algorithms that generally focus on predicting the most detectable peptides we developed a tool that incorporates protein abundance into the peptide prediction algorithm with the aim to determine the detectability of every peptide within a protein. We tested our tool, [prime]Peptide Prediction with Abundance[prime] (PPA), on in-house acquired as well as published datasets from other groups acquired on different instrument platforms. Incorporation of protein abundance into the prediction allows us to assess not only the detectability of all peptides but also whether a peptide of interest is likely to become detectable upon enrichment. We validated the ability of our tool to predict changes in protein detectability with a dilution series of 31 purified proteins at several different concentrations. PPA predicted the concentration dependent peptide detectability in 78% of the cases correctly, demonstrating its utility for predicting the protein enrichment needed to observe a peptide of interest in targeted experiments. This is especially important in the analysis of PTMs. PPA is available as a web-based or executable package that can work with generally applicable defaults or retrained from a pilot MS dataset.