Quantitative proteomics reveals the kinetics of trypsin-catalyzed protein digestion

Trypsin is the popular protease to digest proteins into peptides in shotgun proteomics, but few studies have attempted to systematically investigate the kinetics of trypsin-catalyzed protein digestion in proteome samples. In this study, we applied quantitative proteomics via triplex stable isotope dimethyl labeling to investigate the kinetics of trypsin-catalyzed cleavage. It was found that trypsin cleaves the C-terminal to lysine (K) and arginine (R) residues with higher rates for R. And the cleavage sites surrounded by neutral residues could be quickly cut, while those with neighboring charged residues (D/E/K/R) or proline residue (P) could be slowly cut. In a proteome sample, a huge number of proteins with different physical chemical properties coexists. If any type of protein could be preferably digested, then limited digestion could be applied to reduce the sample complexity. However, we found that protein abundance and other physicochemical properties, such as molecular weight (Mw), grand average of hydropathicity (GRAVY), aliphatic index, and isoelectric point (pI) have no notable correlation with digestion priority of proteins. Graphical Abstract

Sequence logos of four cleavage site types with different kinetics (very fast, fast, slow, and very slow sites)     

Chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family

The ubiquitin (Ub)-proteasome system includes a large family of deubiquitinating enzymes (DUBs). Many members are assigned to this enzyme class by sequence similarity but without evidence for biological activity. A panel of novel DUB-specific probes was generated by a chemical ligation method. These probes allowed identification of DUBs and associated components by tandem mass spectrometry, as well as rapid demonstration of enzymatic activity for gene products whose functions were inferred from primary structure. We identified 23 active DUBs in EL4 cells, including the tumor suppressor CYLD1. At least two DUBs tightly interact with the proteasome 19S regulatory complex. An OTU domain-containing protein, with no sequence homology to any known DUBs, was isolated. We show that this polypeptide reacts with the C terminus of Ub, thus demonstrating DUB-like enzymatic activity for this novel superfamily of proteases.     

Gel-based proteomics reveals potential novel protein markers of ozone stress in leaves of cultivated bean and maize species of Panama

We examined responses of cultivated bean (Phaseolus vulgaris L. cv. IDIAP R-3) and maize (Zea mays L. cv. Guarare 8128) plants exposed to ozone (O(3)) using a leaf injury assessment and proteomics approach. Plants grown for 16 days in greenhouse were transferred to an O(3) chamber and exposed continuously to 0.2 ppm O(3) or filtered pollutant-free air for up to 72 h. CBB-stained gels revealed changes in ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) protein. By Western analysis changes in marker proteins for O(3) damage in leaves by 1-DE were checked. In bean leaves, two superoxide dismutase (SOD) protein (19 and 20 kDa) were dramatically decreased, while ascorbate peroxidase (APX, 25 kDa), small heat shock protein (HSP, 33 kDa), and a naringenin-7-O-methyltransferase (NOMT, 42 kDa) were increased by O(3). In maize leaves, expression levels of catalase (increased), SOD (decreased), and APX (increased) were drastically changed by O(3) depending on the leaf stage, whereas crossreacting HSPs (24 and 30 kDa) and NOMT (41 kDa) proteins were strongly increased in O(3)-stressed younger leaves. These results indicated a clear modulation of oxidative stress-, heat shock-, and secondary metabolism-related proteins by O(3). Finally, 2-DE at 72 h after O(3) exposure revealed changes (induction/suppression) in expression levels of 25 and 12 protein spots in bean and maize leaves, respectively. Out of these, ten and nine nonredundant proteins in bean and maize, respectively, were identified by MS. A novel pathogenesis-related protein 2 may serve as a potential marker for O(3) stress in bean.     

Oxidative protein labeling in mass-spectrometry-based proteomics

Oxidation of proteins and peptides is a common phenomenon, and can be employed as a labeling technique for mass-spectrometry-based proteomics. Nonspecific oxidative labeling methods can modify almost any amino acid residue in a protein or only surface-exposed regions. Specific agents may label reactive functional groups in amino acids, primarily cysteine, methionine, tyrosine, and tryptophan. Nonspecific radical intermediates (reactive oxygen, nitrogen, or halogen species) can be produced by chemical, photochemical, electrochemical, or enzymatic methods. More targeted oxidation can be achieved by chemical reagents but also by direct electrochemical oxidation, which opens the way to instrumental labeling methods. Oxidative labeling of amino acids in the context of liquid chromatography(LC)–mass spectrometry (MS) based proteomics allows for differential LC separation, improved MS ionization, and label-specific fragmentation and detection. Oxidation of proteins can create new reactive groups which are useful for secondary, more conventional derivatization reactions with, e.g., fluorescent labels. This review summarizes reactions of oxidizing agents with peptides and proteins, the corresponding methodologies and instrumentation, and the major, innovative applications of oxidative protein labeling described in selected literature from the last decade.     

Tandem mass spectrometry of multiply phosphorylated forms of a 'histidine-tag' derived from a recombinant protein kinase expressed in bacteria

When a histidine-tagged form of the protein kinase Aurora-2 was expressed in Escherichia coli, the purified product carried four to nine phosphate groups, although many fewer were expected. The amino-terminal tag had the sequence GSSHHHHHHSSGLVPRGSHMK-. Tryptic digestion of the product followed by analysis by liquid chromatography/mass spectrometry (LC/MS) and tandem mass spectrometry (MS/MS) showed that phosphorylation could occur on the five serine residues of the tag. Mono-, bis-, tris-, tetra- and pentaphosphorylated forms of the tag were detected, and their behavior in MS/MS was studied using a quadrupole/time-of-flight mass spectrometer. The MS/MS spectra were dominated by the products of neutral loss events (in 98 Da increments, each equivalent to loss of H3PO4), but sufficient b- and y-type sequence ions were detected to allow the locations of the phosphates to be specified in some cases. The assignment of phosphorylation sites for incompletely phosphorylated forms of the tag peptide was challenging, but it appeared that Ser-10 and Ser-11 of the tag were more likely to be phosphorylated than Ser-2 and Ser-3.     

Chemical proteomics reveals ligustilide targets SMAD3, inhibiting collagen synthesis in aortic endothelial cells

Atherosclerosis is a persistent inflammatory state,while vascular endothelial fibrosis is one of the primary causes of atherosclerosis development.Although ligustilide(Lig) was shown to exert obvious antiatherogenic effects in previous studies,its precise mechanism has not been deeply discussed.In this paper,we designed a Lig-derived photoaffinity labelling(PAL) probe to identify potential therapeutic targets of Lig via chemical proteomics approach.Mothers against decapentaplegic homologue 3(SMAD3),a signal transmitter of transforming growth factor-β(TGF-β) which promotes the development of vascular fibrosis,was identified as a potential target of Lig.Lig suppressed the phosphorylation and nuclear translocation of SMAD3 by blocking the interaction between SMAD3 and TGF-β receptor 1,thereby inhibiting the collagen synthesis process.Hence,developing a novel SMAD3 inhibitor may present a promising therapeutic option for preventing vascular fibrosis.     

Chemistry-based functional proteomics to identify novel deubiquitylating enzymes involved in viral infection

Ubiquitylation is a reversible post-translational modification pathway that regulates a variety of cellular processes including protein degradation and trafficking, intracellular localization, DNA repair, immune response and cellcycle progression. Deubiquitylating enzymes (DUBs) can remove the ubiquitin from the modified proteins and reverse the ubiquitylation-induced biological processes; hence it isn't hard to understand that viral pathogens take advantage of the host cell ubiquitin system through disturbing DUBs, for infection and replication. Although accumulated virus-related DUBs have been defined, but how viruses regulate their expression and activities is poor understand because of limitation of technologies. Recently, chemistry-based functional proteomics, which can not only monitor the alteration of abundance but also changes in activity of enzymes, was used to study the function of DUBs involved in virus infection and held much promise. Theses works suggest that chemistry-based functional proteomics is a potent strategy for high throughput screening of virus-related DUBs and exploring their roles in virus infection.     

Tandem mass spectrometry methods for definitive protein identification in proteomics research

Optimized procedures have been developed for the addition of sulfonic acid groups to the N-termini of low-level peptides. These procedures have been applied to peptides produced by tryptic digestion of proteins that have been separated by two-dimensional (2-D) gel electrophoresis. The derivatized peptides were sequenced using matrix-assisted laser desorption/ionization (MALDI) post-source decay (PSD) and electrospray ionization-tandem mass spectrometry methods. Reliable PSD sequencing results have been obtained starting with sub-picomole quantities of protein. We estimate that the current PSD sequencing limit is about 300 fmol of protein in the gel. The PSD mass spectra of the derivatized peptides usually allow much more specific protein sequence database searches than those obtained without derivatization. We also report initial automated electrospray ionization-tandem mass spectrometry sequencing of these novel peptide derivatives. Both types of tandem mass spectra provide predictable fragmentation patterns for arginine-terminated peptides. The spectra are easily interpreted de novo, and they facilitate error-tolerant identification of proteins whose sequences have been entered into databases.     

Restricted access chromatographic sample preparation of low mass proteins expressed in human fibroblast cells for proteomics analysis

Two-dimensional electrophoresis and modern image analysis systems have made it possible to study protein expression and regulation of proteins in biological systems. Proteins in the molecular mass region of 20-120 kDa are well investigated and described. However, proteins with masses below 20 kDa are the least investigated as they are rarely seen on 2D-PAGE due to fast migrations in the electric field and lack of staining efficiency. This paper describes a technique that enriches proteins in the lower mass region using solid-phase extraction. The purification step is carried out using C18 functionalised "restricted access" affinity chromatography whereby simultaneous trace enrichment and sample clean up is achieved. In this study expression patterns of TGF-beta stimulated and non-stimulated fibroblasts were compared after the solid-phase fractionation procedure. An increased expression pattern was obtained whereby 400 protein spots could be detected by image analysis in the <20-kDa region. Out of these, specific regulations of 14 spots were found by quantitative image analysis and spots of interest were identified with MALDI TOF-MS. The regulated and identified proteins were triosephosphate isomerase, cofilin and heat shock 27-kDa protein.     

ProteinLasso: A Lasso regression approach to protein inference problem in shotgun proteomics

Protein inference is an important issue in proteomics research. Its main objective is to select a proper subset of candidate proteins that best explain the observed peptides. Although many methods have been proposed for solving this problem, several issues such as peptide degeneracy and one-hit wonders still remain unsolved. Therefore, the accurate identification of proteins that are truly present in the sample continues to be a challenging task.Based on the concept of peptide detectability, we formulate the protein inference problem as a constrained Lasso regression problem, which can be solved very efficiently through a coordinate descent procedure. The new inference algorithm is named as ProteinLasso, which explores an ensemble learning strategy to address the sparsity parameter selection problem in Lasso model. We test the performance of ProteinLasso on three datasets. As shown in the experimental results, ProteinLasso outperforms those state-of-the-art protein inference algorithms in terms of both identification accuracy and running efficiency. In addition, we show that ProteinLasso is stable under different parameter specifications. The source code of our algorithm is available at: http://sourceforge.net/projects/proteinlasso.     

Progress in protein structural class prediction and its impact to bioinformatics and proteomics

The structural class is an important attribute used to characterize the overall folding type of a protein or its domain. Since the concept of protein structural class was developed about 3 decades ago based on a visual inspection of polypeptide chain topologies in a dataset of only 31 gloular proteins, the number of structure-known proteins has been increased rapidly. For example, as of 12-July-2005, the entries deposited into RCSB PDB Protein Data Bank for proteins, peptides, and viruses whose 3-dimensional structures were determined by X-ray and NMR techniques have been increased to 28,920. To properly cover more and more structure-known proteins, some modification and expansion from the original structural classification scheme have been developed. Meanwhile, many different approaches have been proposed for predicting the structural class of proteins. In this review, the new classification schemes are briefly introduced. The attention is focused on the progress in structural class prediction and its impact in stimulating the development of identifying the other attributes of proteins. It is interesting to point out that the development of the latter has actually in turn greatly enriched the power of the former. Also, some promising approaches for the further development of protein structural class prediction are also addressed.     

Emerging methods in proteomics: top-down protein characterization by multistage tandem mass spectrometry

"Top-down" mass spectrometry methods have emerged as an attractive alternative to conventional "bottom-up" approaches for the comprehensive characterization of co- and post-translational protein modifications. Here we present a brief overview of current strategies employed for top-down protein characterization and discuss the key technical challenges and solutions associated with their implementation on a range of mass spectrometry instrument platforms. For more specific details regarding the individual strategies described herein, interested readers are referred to the references cited at the end of this article.     

Advances in the analysis of dynamic protein complexes by proteomics and data processing

Signal transduction governs virtually every cellular function of multicellular organisms, and its deregulation leads to a variety of diseases. This intricate network of molecular interactions is mediated by proteins that are assembled into complexes within individual signaling pathways, and their composition and function is often regulated by different post-translational modifications. Proteomic approaches are commonly used to analyze biological complexes and networks, but often lack the specificity to address the dynamic and hence transient nature of the interactions and the influence of the multiple post-translational modifications that govern these processes. Here we review recent developments in proteomic research to address these limitations, and discuss several technologies that have been developed for this purpose. The synergy between these proteomic and computational tools, when applied together with global methods to the analysis of individual proteins, complexes and pathways, may allow researchers to unravel the underlying mechanisms of signaling networks in greater detail than previously possible.     

Biolabel-led research pattern reveals serum profile in rats after treatment with Herba Lysimachiae: Combined analysis of metabonomics and proteomics

In traditional Chinese medicine, Herba Lysimachiae (HL) is mainly used to treat rheumatic arthralgia. Current pharmacological studies also showed that HL has therapeutic potential for synovial diseases. HL is an oral drug, whose compounds need to enter the blood circulation before reaching the injured tissue, thus potentially causing activity or toxicity to the blood system. In this study, the biolabel-led research pattern was used to analyze the serum profile after HL intervention, based on which the safety and efficacy of HL were explored. Metabonomics and proteomics were combined to analyze the biolabels responsible for the interventions of HL in serum. Bioinformatics databases were used to screen for the material basis that may interfere with biolabels. Omics analysis showed that differentially expressed proteins (19) and metabolites (5) were identified and considered as the potential biolabels, which were involved in 8 biochemical processes (platelet activation and aggregation, blood glucose release, immune and inflammatory regulation, oxidative stress, endoplasmic reticulum stress, tumor progression, blood pressure regulation, and uric acid synthesis). Thirty-one compounds may be the material basis to interfere with 11 biolabels. The present research reveals that the potential activities and toxicities of HL can be explored based on the biolabel-led research pattern.     

Production and characterization of an antibody specific for a novel protein serine/threonine kinase, MPK38, highly expressed in hematopoietic cells

We report an antibody that selectively recognizes MPK38, a new protein serine/threonine kinase closely related to the SNF1 serine/threonine kinase family. This antibody recognized a region of the N-terminal kinase catalytic domain and part of the remaining C-terminal portion and was sensitive enough to detect a 72-kDa recombinant MPK38 in insect cells by Western blotting. Immunoblot analysis showed that the recombinant MPK38 was expressed in a time-dependent manner and reached a maximum after 48 h postinfection. In addition, the immune complex kinase assay revealed that the recombinant and endogenous MPK38 protein autophosphorylated in vitro. Phosphoamino acid analysis of autophosphorylated MPK38 protein showed that the phosphorylation was exclusively on serine and threonine residues, suggesting that MPK38 is a protein serine/threonine kinase. Thus, this antibody could be helpful for elucidating the biological functions of MPK38 in the MPK38-expressing cells.