Systematic Evaluation of Immobilized Trypsin‐Based Fast Protein Digestion for Deep and High‐Throughput Bottom‐Up Proteomics

Immobilized trypsin (IM) has been recognized as an alternative to free trypsin (FT) for accelerating protein digestion 30 years ago. However, some questions of IM still need to be answered. How does the solid matrix of IM influence its preference for protein cleavage and how well can IM perform for deep bottom‐up proteomics compared to FT? By analyzing Escherichia coli proteome samples digested with amine or carboxyl functionalized magnetic bead–based IM (IM‐N or IM‐C) or FT, it is observed that IM‐N with the nearly neutral solid matrix, IM‐C with the negatively charged solid matrix, and FT have similar cleavage preference considering the microenvironment surrounding the cleavage sites. IM‐N (15 min) and FT (12 h) both approach 9000 protein identifications (IDs) from a mouse brain proteome. Compared to FT, IM‐N has no bias in the digestion of proteins that are involved in various biological processes, are located in different components of cells, have diverse functions, and are expressed in varying abundance. A high‐throughput bottom‐up proteomics workflow comprising IM‐N‐based rapid protein cleavage and fast CZE‐MS/MS enables the completion of protein sample preparation, CZE‐MS/MS analysis, and data analysis in only 3 h, resulting in 1000 protein IDs from the mouse brain proteome.     

Increased proteome coverage by combining PAGE and peptide isoelectric focusing: Comparative study of gel‐based separation approaches

The in‐depth analysis of complex proteome samples requires fractionation of the sample into subsamples prior to LC‐MS/MS in shotgun proteomics experiments. We have established a 3D workflow for shotgun proteomics that relies on protein separation by 1D PAGE, gel fractionation, trypsin digestion, and peptide separation by in‐gel IEF, prior to RP‐HPLC‐MS/MS. Our results show that applying peptide IEF can significantly increase the number of proteins identified from PAGE subfractionation. This method delivers deeper proteome coverage and provides a large degree of flexibility in experimentally approaching highly complex mixtures by still relying on protein separation according to molecular weight in the first dimension.     

Reference proteome of highly purified human Th1 cells reveals strong effects on metabolism and protein ubiquitination upon differentiation

The differentiation of human CD4⁺ T cells into T helper cell subtypes and regulatory T cells is crucial to the immune response. Among subtypes, Th1 cells are dominant, representing approximately 50% of all lymphocytes. Thus far, most global proteomic studies have used only partially purified T helper cell subpopulations and/or have employed artificial protocols for inducing specific T helper cell subtypes and/or used gel‐based approaches. These studies have shed light on molecular details of certain aspects of the proteome; nevertheless a global analysis of high purity primary naïve and Th1 cells by LC‐MS/MS is required to provide a reference dataset for proteome‐based T cell subtype characterization. The utilization of highly purified Th1 cells for a global proteome assessment and the bioinformatic comparison to naïve cells reveals changes in cell metabolism and the ubiquitination pathway upon T cell differentiation. All MS data have been deposited in the ProteomeXchange with identifier PXD001066 ( http://proteomecentral.proteomexchange.org/dataset/PXD001066 ).     

Activity profiling of platelets by chemical proteomics

Chemical proteomics or activity based proteomics is a functional proteomics technology where molecular probes are used to target a selective group of functionally related proteins. Its emergence has enabled specific targeting of subproteomes, overcoming the limitations in dynamic range of traditional large‐scale proteomics experiments. Using a chemical proteomics strategy, we attempt to differentially profile the nucleotide‐binding proteome of active and resting platelets. We apply an affinity chromatography protocol using immobilized adenosine triphosphate, cyclic adenosine monophosphate, and cyclic guanosine monophosphate. The specificity of the immobilized nucleotides was demonstrated by competitive assays and by immunoblotting. LC coupled MS/MS was applied to identify the proteins recovered by our chemical proteomics strategy. When compared to a standard set of platelet lysate proteins, we confirmed that enrichment for nucleotide‐binding proteins was indeed taking place. Finally, by employing label‐free MS‐based comparative quantification, we found a small number of platelet proteins that show statistically significant difference between the active and resting nucleotide‐binding proteome.     

Purification of cybrids by fluorescence-activated cell sorting

A general method to isolate and purify substantial numbers of viable cybrids from cultured mammalian cells immediately following cytoplast-cell fusion is described. This method uses cytoplasts whose mitochondria are selectively stained in vivo by the cationic fluorescent rhodamine dye, rhodamine 123. Large numbers of highly purified, rhodamine-stained cytoplasts are fused to appropriate recipient cell lines and then the fusion mixture is sorted based on forward angle scatter and fluorescence parameters. Plating the positively sorted population in culture for as short as 12 h eliminates contaminating cytoplasts which, lacking a nucleus, are unable to adhere or survive. The resultant population, based on an analysis of genetic markers, is 75-100% cybrids, an enrichment of 1000- to 10,000-fold over the initial fusion mixture. Cybrids purified by cell sorting may be useful for detailed molecular studies of mitochondrial DNA gene expression and in the specific induction of new mitochondrial DNA mutants.     

Combined analysis of transcriptome and proteome data as a tool for the identification of candidate biomarkers in renal cell carcinoma

Results obtained from expression profilings of renal cell carcinoma using different “ome”‐based approaches and comprehensive data analysis demonstrated that proteome‐based technologies and cDNA microarray analyses complement each other during the discovery phase for disease‐related candidate biomarkers. The integration of the respective data revealed the uniqueness and complementarities of the different technologies. While comparative cDNA microarray analyses though restricted to up‐regulated targets largely revealed genes involved in controlling gene/protein expression (19%) and signal transduction processes (13%), proteomics/PROTEOMEX‐defined candidate biomarkers include enzymes of the cellular metabolism (36%), transport proteins (12%), and cell motility/structural molecules (10%). Candidate biomarkers defined by proteomics and PROTEOMEX are frequently shared, whereas the sharing rate between cDNA microarray and proteome‐based profilings is limited. Putative candidate biomarkers provide insights into their cellular (dys)function and their diagnostic/prognostic value but still warrant further validation in larger patient numbers. Based on the fact that merely three candidate biomarkers were shared by all applied technologies, namely annexin A4, tubulin α‐1A chain, and ubiquitin carboxyl‐terminal hydrolase L1, the analysis at a single hierarchical level of biological regulation seems to provide only limited results thus emphasizing the importance and benefit of performing rather combinatorial screenings which can complement the standard clinical predictors.     

Characterisation of the nuclear proteome of a dehydration‐sensitive cultivar of chickpea and comparative proteomic analysis with a tolerant cultivar

Water deficit or dehydration hampers plant growth and development, and shrinks harvest size of major crop species worldwide. Therefore, a better understanding of dehydration response is the key to decipher the regulatory mechanism of better adaptation. In recent years, nuclear proteomics has become an attractive area of research, particularly to study the role of nucleus in stress response. In this study, a proteome of dehydration‐sensitive chickpea cultivar (ICCV‐2) was generated from nuclei‐enriched fractions. The LC‐MS/MS analysis led to the identification of 75 differentially expressed proteins presumably associated with different metabolic and regulatory pathways. Nuclear localisation of three candidate proteins was validated by transient expression assay. The ICCV‐2 proteome was then compared with that of JG‐62, a tolerant cultivar. The differential proteomics and in silico analysis revealed cultivar‐specific differential expression of many proteins involved in various cellular functions. The differential tolerance could be attributed to altered expression of many structural proteins and the proteins involved in stress adaptation, notably the ROS catabolising enzymes. Further, a comprehensive comparison on the abiotic stress‐responsive nuclear proteome was performed using the datasets published thus far. These findings might expedite the functional determination of the dehydration‐responsive proteins and their prioritisation as potential molecular targets for better adaptation.     

Proteomics made more accessible

MS‐based proteomics is a bioinformatic‐intensive field. Additionally, the instruments and instrument‐related and analytic software are expensive. Some free Internet‐based proteomics tools have gained wide usage, but there have not been any single bioinformatic framework that in an easy and intuitive way guided the user through the whole process from analyses to submission. Together, these factors may have limited the expansion of proteomics analyses, and also the secondary use (reanalyses) of proteomic data. Vaudel et al. (Proteomics 2014, 14, 1001–1005) are now describing their Compomics framework that guides the user through all the main steps, from the database generation, via the analyses and validation, and through the submission process to PRIDE, a proteomic data bank. Vaudel et al. partly base the framework on tools that they have developed themselves, and partly they are integrating other freeware tools into the workflow. One of the most interesting aspects with the Compomics framework is the possibility of extending MS‐based proteomics outside the MS laboratory itself. With the Compomics framework, any laboratory can handle large amounts of proteomic data, thereby facilitating collaboration and in‐depth data analyses. The described software also opens the potential for any laboratory to reanalyze data deposited in PRIDE.     

The human oligodendrocyte proteome

Myelination of the CNS is performed by oligodendrocytes (OLs), which have been implicated in brain disorders, such as multiple sclerosis and schizophrenia. We have used the human oligodendroglial cell line MO3.13 to establish an OL reference proteome database. Proteins were prefractionationated by SDS‐PAGE and after in‐gel digestion subjected to nanoflow LC‐MS analysis. Approximately 11 600 unique peptides were identified and, after stringent filtering, resulted in 2290 proteins representing nine distinct biological processes and various molecular classes and functions. OL‐specific proteins, such as myelin basic protein (MBP) and 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP), as well as other proteins involved in multiple sclerosis and schizophrenia were also identified and are discussed. Proteins of this dataset have also been classified according to their chromosomal origin for providing useful data to the Chromosome‐centric Human Proteome Project (C‐HPP). Given the importance of OLs in the etiology of demyelinating and oligodendrogial disorders, the MO3.13 proteome database is a valuable data resource. The MS proteomics data have been deposited to the ProteomeXchange with identifier PXD000263 ( http://proteomecentral.proteomexchange.org/dataset/PXD000263 ).     

Proteomics analysis of BHK‐21 cells infected with a fixed strain of rabies virus

Rabies is a neurotropic virus that causes a life threatening acute viral encephalitis. The complex relationship of rabies virus (RV) with the host leads to its replication and spreading toward the neural network, where viral pathogenic effects appeared as neuronal dysfunction. In order to better understand the molecular basis of this relationship, a proteomics study on baby hamster kidney cells infected with challenge virus standard strain of RV was performed. This cell line is an in vitro model for rabies infection and is commonly used for viral seed preparation. The direct effect of the virus on cellular protein machinery was investigated by 2‐DE proteome mapping of infected versus control cells followed by LC‐MS/MS identification. This analysis revealed significant changes in expression of 14 proteins, seven of these proteins were viral and the remaining were host proteins with different known functions: cytoskeletal (capping protein, vimentin), anti‐oxidative stress (superoxide dismutase), regulatory (Stathmin), and protein synthesis (P0). Despite of limited changes appeared upon rabies infection, they present a set of interesting biochemical pathways for further investigation on viral‐host interaction.     

Proteomic profiling of distinct clonal populations of bone marrow mesenchymal stem cells

Mesenchymal stem cells (MSCs) have attracted immense research interest in the field of regenerative medicine due to their ability to be cultured for successive passages and multi‐lineage differentiation. The molecular mechanisms governing MSC self‐renewal and differentiation remain largely unknown. The development of sophisticated techniques, in particular clinical proteomics, has enabled researchers in various fields to identify and characterize cell specific biomarkers for therapeutic purposes. This study seeks to understand the cellular and sub‐cellular processes responsible for the existence of stem cell populations in bone marrow samples by revealing the whole cell proteome of the clonal cultures of bone marrow‐derived MSCs (BMSCs). Protein profiling of the MSC clonal populations was conducted by Two‐Dimensional Liquid Chromatography/Matrix‐Assisted Laser Desorption/Ionisation (MALDI) Mass Spectrometry (MS). A total of 83 proteins were identified with high confidence of which 11 showed differential expression between subpopulations, which included cytoskeletal and structural proteins, calcium binding proteins, cytokinetic proteins, and members of the intermediate filament family. This study generated a proteome reference map of BMSCs from the clonal populations, which will be valuable to better understand the underlying mechanism of BMSC self‐renewal and differentiation. J. Cell. Biochem. 106: 776–786, 2009. © 2009 Wiley‐Liss, Inc.     

Proteomic exploration of the impacts of pomegranate fruit juice on the global gene expression of prostate cancer cell

Prostate cancer has been known to be the second highest cause of death in cancer among men. Pomegranate is rich in polyphenols with the potent antioxidant activity and inhibits cell proliferation, invasion, and promotes apoptosis in various cancer cells. This study demonstrated that pomegranate fruit juice could effectively hinder the proliferation of human prostate cancer DU145 cell. The results of apoptotic analyses implicated that fruit juice might trigger the apoptosis in DU145 cells via death receptor signaling and mitochondrial damage pathway. In this study, we exploited 2DE‐based proteomics to compare nine pairs of the proteome maps collected from untreated and treated DU145 cells to identify the differentially expressed proteins. Comparative proteomics indicated that 11 proteins were deregulated in affected DU145 cells with three upregulated and eight downregulated proteins. These dys‐regulated proteins participated in cytoskeletal functions, antiapoptosis, proteasome activity, NF‐κB signaling, cancer cell proliferation, invasion, and angiogenesis. Western immunoblotting were implemented to confirm the deregulated proteins and the downstream signaling proteins. The analytical results of this study help to provide insight into the molecular mechanism of inducing prostate cancer cell apoptosis by pomegranate fruit juice and to develop a novel mechanism‐based chemopreventive strategy for prostate cancer.     

Improving Protein Detection Confidence Using SWATH‐Mass Spectrometry with Large Peptide Reference Libraries

Protein quantification using data‐independent acquisition methods such as SWATH‐MS most commonly relies on spectral matching to a reference MS/MS assay library. To enable deep proteome coverage and efficient use of existing data, in silico approaches have been described to use archived or publicly available large reference spectral libraries for spectral matching. Since implicit in the use of larger libraries is the increasing likelihood of false‐discoveries, new workflows are needed to ensure high confidence in protein matching under these conditions. We present a workflow which introduces a range of filters and thresholds aimed at increasing confidence that the resulting proteins are reliably detected and their quantitation is consistent and reproducible. We demonstrated the workflow using extended libraries with SWATH data from human plasma samples and yeast‐spiked human K562 cell lysate digest.     

Time‐resolved quantitative proteome profiling of host–pathogen interactions: The response of Staphylococcus aureus RN1HG to internalisation by human airway epithelial cells

Staphylococcus aureus is a versatile Gram‐positive pathogen that gains increasing importance due to the rapid spreading of resistances. Functional genomics technologies can provide new insights into the adaptational network of this bacterium and its response to environmental challenges. While functional genomics technologies, including proteomics, have been extensively used to study these phenomena in shake flask cultures, studies of bacteria from in vivo settings lack behind. Particularly for proteomics studies, the major bottleneck is the lack of sufficient proteomic coverage for low numbers of cells. In this study, we introduce a workflow that combines a pulse‐chase stable isotope labelling by amino acids in cell culture approach with high capacity cell sorting, on‐membrane digestion, and high‐sensitivity MS to detect and quantitatively monitor several hundred S. aureus proteins from a few million internalised bacteria. This workflow has been used in a proof‐of‐principle experiment to reveal changes in levels of proteins with a function in protection against oxidative damage and adaptation of cell wall synthesis in strain RN1HG upon internalisation by S9 human bronchial epithelial cells.     

The challenge of the proteome dynamic range and its implications for in‐depth proteomics

The dynamic range of the cellular proteome approaches seven orders of magnitude—from one copy per cell to ten million copies per cell. Since a proteome's abundance distribution represents a nearly symmetric bell‐shape curve on the logarithmic copy number scale, detection of half of the expressed cellular proteome, i.e. approximately 5000 proteins, should be a relatively straightforward task with modern mass spectrometric instrumentation that exhibits four orders of magnitude of the dynamic range, while deeper proteome analysis should be progressively more difficult. Indeed, metaanalysis of 15 recent papers that claim detection of >5000 protein groups reveals that the half‐proteome analyses currently requires ≈5 h of chromatographic separation, while deeper analyses yield on average ≤20 new proteins per hour of chromatographic gradient. Therefore, a typical proteomics experiment consists of a “high‐content” part, with the detection rate of approximately 1000 proteins/h, and a “low‐content” tail with much lower rate of discovery and respectively, lower cost efficiency. This result calls for disruptive innovation in deep proteomics analysis.     

Proteomic analysis of calcium oxalate monohydrate crystal-induced cytotoxicity in distal renal tubular cells

Calcium oxalate monohydrate (COM) is the major crystalline component found in kidney stones and its adhesion to renal tubular cells provokes tubular injury, which in turn enhances COM crystal adhesion. However, COM-induced toxic effects in these tubular cells remain largely unknown. We performed a proteomics study to characterize changes in the cellular proteome in MDCK distal renal tubular cells after an exposure to high-dose (1000 microg/mL) COM crystals for 48 h, at which percentage of cell death was significantly increased. Proteins were extracted from MDCK cells cultured with COM-containing or COM-free medium ( n = 5 individual flasks per group), resolved in individual 2-D gels, and stained with SYPRO Ruby fluorescence dye. Quantitative and statistical analyses revealed 53 proteins whose abundance levels were altered (25 were increased, whereas other 28 were decreased) by COM-induced toxicity. Among these, 50 were successfully identified by quadrupole time-of-flight (Q-TOF) mass spectrometry (MS) and/or tandem MS (MS/MS) analyses. The proteomic data were clearly confirmed by 2-D Western blot analysis. While three chaperones (GRP78, Orp150 and Hsp60) were increased, other proteins involved in protein biosynthesis, ATP synthesis, cell cycle regulator, cellular structure, and signal transduction were decreased. These data provide some novel mechanistic insights into the molecular mechanisms of COM crystal-induced tubular toxicity.