Differentially isotope-coded N-terminal protein sulphonation: combining protein identification and quantification

Most proteomic labelling technologies intend to improve protein quantification and/or facilitate (de novo) peptide sequencing. We present here a novel stable-isotope labelling method to simultaneously identify and quantify protein components in complex mixtures by specifically derivatizing the N-terminus of proteins with 4-sulphophenyl isothiocyanate (SPITC). Our approach combines protein identification with quantification through differential isotope-coded labelling at the protein N-terminus prior to digestion. The isotope spacing of 6 Da (unlabelled vs. six-fold 13C-labelled tag) between derivatized peptide pairs enables the detection on different MS platforms (MALDI and ESI). Optimisation of the reaction conditions using SPITC was performed on three model proteins. Improved detection of the N-terminally derivatized peptide compared to the native analogue was observed in negative-ion MALDI-MS. Simpler fragmentation patterns compared to native peptides facilitated protein identification. The 13C-labelled SPITC resulted in convenient peptide pair spacing without isotopic overlap and hence facilitated relative quantification by MALDI-TOF/TOF and LC-ESI-MS/MS. The combination of facilitated identification and quantification achieved by differentially isotope-coded N-terminal protein tagging with light/heavy SPITC represents, to our knowledge, a new approach to quantitative proteomics.     

Proteome analysis of secreted proteins during osteoclast differentiation using two different methods: two-dimensional electrophoresis and isotope-coded affinity tags analysis with two-dimensional chromatography

Bone is maintained by two cell types, bone-forming osteoblasts and bone-resorbing osteoclasts. Osteoblasts express two factors, osteoprotegerin and receptor activator of NF-kappaB ligand (RANKL), inhibiting and promoting osteoclast differentiation, respectively. In contrast, modulators of bone resorption expressed by osteoclasts have not been so well studied enough. In the present study, we demonstrate proteome analysis of secreted proteins during osteoclast differentiation to elucidate the molecular mechanism of bone resorption and bone remodeling. To achieve this objective, we chose RAW264.7 cells with RANKL as a homogeneous osteoclast differentiation model and used two methods, two-dimensional gel electrophoresis (2-DE) and isotope-coded affinity tags (ICAT) analysis with two-dimensional liquid chromatography. We found 23 spots in 2-DE and 19 proteins in ICAT analysis which were expressed differently during osteoclast differentiation. These two methods gave us closely related but different information about proteins, suggesting they are complementary or at least supplementary methods at present. Cathepsins, osteopontin, legumain, macrophage inflammatory protein-1alpha, and other proteins were observed as up- or down-regulated proteins and are discussed in the context of osteoclast differentiation and bone resorption. In addition to confirming previous observations, this study indicates novel proteins related to osteoclast differentiation which are potential therapeutic targets for the treatment of bone diseases, such as osteoporosis.     

Identification of membrane-associated proteins from Campylobacter jejuni strains using complementary proteomics technologies

Campylobacter jejuni is the leading cause of food- and water-borne illness world-wide. The membrane-associated proteome of a recent C. jejuni gastrointestinal isolate (JHH1) was generated by sodium carbonate precipitation and ultracentrifugation followed by 2-DE and MALDI-TOF MS as well as 2-DLC (strong cation exchange followed by RP chromatography) of trypsin digests coupled to MS/MS (2-DLC/MS/MS). 2-DE/MS identified 77 proteins, 44 of which were predicted membrane proteins, while 2-DLC/MS/MS identified 432 proteins, of which 206 were predicted to be membrane associated. A total of 453 unique proteins (27.4% of the C. jejuni theoretical proteome), including 187 bona fide membrane proteins were identified in this study. Membrane proteins were also compared between C. jejuni JHH1 and ATCC 700297 to identify factors potentially associated with increased gastrointestinal virulence. We identified 28 proteins that were significantly (>two-fold) more abundant in, or unique to, JHH1, including eight proteins involved in chemotaxis signal transduction and flagellar motility, the amino acid-binding surface antigens CjaA and CjaC, and four outer membrane proteins (OMPs) of unknown function (Cj0129c, Cj1031, Cj1279c, and Cj1721c). Immunoblotting using convalescent patient sera generated post-gastrointestinal infection revealed 13 (JHH1) and 12 (ATCC 700297) immunoreactive proteins. These included flagellin (FlaA) and CadF as well as Omp18, Omp50, Cj1721c, PEB1A, PEB2, and PEB4A. This study provides a comprehensive analysis of membrane-associated proteins from C. jejuni.     

ICPLQuant – A software for non‐isobaric isotopic labeling proteomics

The main goal of many proteomics experiments is an accurate and rapid quantification and identification of regulated proteins in complex biological samples. The bottleneck in quantitative proteomics remains the availability of efficient software to evaluate and quantify the tremendous amount of mass spectral data acquired during a proteomics project. A new software suite, ICPLQuant, has been developed to accurately quantify isotope‐coded protein label (ICPL)‐labeled peptides on the MS level during LC‐MALDI and peptide mass fingerprint experiments. The tool is able to generate a list of differentially regulated peptide precursors for subsequent MS/MS experiments, minimizing time‐consuming acquisition and interpretation of MS/MS data. ICPLQuant is based on two independent units. Unit 1 performs ICPL multiplex detection and quantification and proposes peptides to be identified by MS/MS. Unit 2 combines MASCOT MS/MS protein identification with the quantitative data and produces a protein/peptide list with all the relevant information accessible for further data mining. The accuracy of quantification, selection of peptides for MS/MS‐identification and the automated output of a protein list of regulated proteins are demonstrated by the comparative analysis of four different mixtures of three proteins (Ovalbumin, Horseradish Peroxidase and Rabbit Albumin) spiked into the complex protein background of the DGPF Proteome Marker.     

Quantitative analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced proteome alterations in 5L rat hepatoma cells using isotope-coded protein labels

In an effort to contribute to a better understanding of the hepatic toxicity of the ubiquitous environmental pollutant and hepatocarcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a comprehensive quantitative proteome analysis was performed on 5L rat hepatoma cells exposed to 1 nM TCDD for 8 h. Changes in the abundances of individual protein species in TCDD-treated cells as compared to untreated cells were analysed using the nongel-based isotope-coded protein label (ICPL) method [Schmidt, A., Kellermann, J., Lottspeich, F., Proteomics 2005, 5, 4-15]. 89 proteins were identified as up- or down-regulated by TCDD. For the majority of the altered proteins, an impact of TCDD on their abundance had not been known before. Due to the physicochemical properties or the translational regulation of a large number of the affected proteins, their alteration would have escaped detection by gel-based methods for proteome analysis and by standard mRNA expression profiling, respectively. The identified proteins with TCDD-altered abundance include several proteins implicated in cell cycle regulation, growth factor signalling and the control of apoptosis. The results thus provide new starting-points for the investigation of specific aspects of the toxicity and carcinogenicity of dioxin in liver.     

A multidimensional proteomic approach to identify hypertrophy-associated proteins

Left ventricular hypertrophy (LVH) is a leading cause of congestive heart failure. The exact mechanisms that control cardiac growth and regulate the transition to failure are not fully understood, in part due to the lack of a complete inventory of proteins associated with LVH. We investigated the proteomic basis of LVH using the transverse aortic constriction model of pressure overload in mice coupled with a multidimensional approach to identify known and novel proteins that may be relevant to the development and maintenance of LVH. We identified 123 proteins that were differentially expressed during LVH, including LIM proteins, thioredoxin, myoglobin, fatty acid binding protein 3, the abnormal spindle-like microcephaly protein (ASPM), and cytoskeletal proteins such as actin and myosin. In addition, proteins with unknown functions were identified, providing new directions for future research in this area. We also discuss common pitfalls and strategies to overcome the limitations of current proteomic technologies. Together, the multidimensional approach provides insight into the proteomic changes that occur in the LV during hypertrophy.     

18O-labeling quantitative proteomics using an ion trap mass spectrometer

We describe a method for simultaneous identification and quantitation of proteins within complex mixtures. The method consists of 18O-labeling, a simple stable isotope-coding that requires merely enzymatic digestion in 18O-water, in combination with a capillary-liquid chromatography electrospray ion-trap mass spectrometer. In a separate experiment using the same sample and a spike test, we demonstrate that the difference ration was calculated accurately using the 18O-labeling method even if the protein was part of a complex mixture. Our data also suggest that the accuracy of the quantitation can be improved by averaging the difference ratios of several peptides. In comparing our method with the isotope-coded affinity tag (ICAT) method, we show that the 18O-labeling method has the advantages of better recovery and fewer isotope effects. Therefore, the 18O-labeling method is a powerful tool for large-scale proteomics applications.     

Technical strategies to reduce the amount of "false significant" results in quantitative proteomics

When the p-value is set at <0.05 in statistical group comparisons, a 5% rate of "false significant" results is expected. In order to test the reliability of our 2-DE method, we loaded each of 24 gels with equal-sized samples (200 mug protein from pooled rat brain, pH 4-7, stained with ruthenium fluorescent stain for visualization) and statistically compared the first 12 gels with the last 12. In numerous experiments the rate of significant differences found far exceeded 5%. Several factors were identified as causing the following rates of false significant differences in spot intensities: (i) running samples in two different 2-DE runs (42%), (ii) running second dimension gels produced in two different gel casters (16%), (iii) normalizing the entire gel instead of separately normalizing several different gel zones (11%), (iv) using IPG strips from different packages (19%), (v) dividing the whole sample into subgroups during software analysis (9%). After controlling for all these factors, the rates of "false positive" results in our experiments were regularly reduced to approximately 5%. This is an indispensable prerequisite for avoiding too high a rate of false positive results in experiments in which different subgroups are compared statistically.     

Comparison of extraction procedures for proteome analysis of Streptococcus pneumoniae and a basic reference map

Streptococcus pneumoniae is an important human pathogen causing life-threatening invasive diseases such as pneumonia, meningitis and bacteraemia. Despite major advances in our understanding of pneumococcal mechanisms of pathogenicity obtained through genomic studies very little has been achieved on the characterisation of the proteome of this pathogen. The highly complex structure of its cell envelope particularly amongst the various capsular forms enables the cell to resist lysis by conventional mechanical methods. It is therefore highly desirable to develop a cellular lysis and protein solubilisation procedure that minimises protein losses and allows for maximum possible coverage of the proteome of S. pneumoniae. Here we have utilised various combinations of mechanical or enzymatic cell lysis with two protein solubilisation mixtures urea/CHAPS-based mixture or SDS/DTT-based mixture in order to achieve best quality protein profiles using two proteomic technologies surface-enhanced laser desorption ionisation (SELDI) TOF MS and 2-DE. While urea/CHAPS-based mixture combined with freeze/thawing provided enough material for good-quality SELDI TOF MS fingerprints, a combination of mechanical, enzymatic and chemical lysis was needed to be used to successfully extract the desired protein content for 2-DE analysis. The methods chosen were also assessed for reproducibility and tested on various capsular types of S. pneumoniae. As a result, good-quality and reproducible profiles were created using various ProteinChip arrays and more than 800 protein spots were separated on a single 2-D gel of S. pneumoniae. Twenty-five of the most abundant protein spots were identified using LC/MS/MS to create a reference map of S. pneumoniae. The proteins identified included glycolytic enzymes such as glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, enolase etc. Several fermentation enzymes were also present including two of the components of the arginine deiminase system. Proteins involved in protein synthesis, such as translation factors and ribosomal proteins, as well as several chaperone proteins were also identified.     

A novel approach to spot detection for two-dimensional gel electrophoresis images using pixel value collection

Separation of complex mixtures of proteins by two-dimensional gel electrophoresis (2-DE) is a fundamental component of current proteomic technology. Quantitative analysis of the images generated by digitization of such gels is critical for the identification of alterations in protein expression within a given biological system. Despite the availability of several commercially available software packages designed for this purpose, image analysis is extremely resource intensive, subjective and remains a major bottleneck. In addition to reducing throughput, the requirement for manual intervention results in the introduction of operator subjectivity, which can limit the statistical significance of the numerical data generated. A key requirement of image analysis is the accurate definition of protein spot boundaries using a suitable method of image segmentation. We describe a method of spot detection applicable to 2-DE image files using a segmentation method involving pixel value collection via serial analysis of the image through its range of density levels. This algorithm is reproducible, sensitive, accurate and primarily designed to be automatic, removing operator subjectivity. Furthermore, it is believed that this method may offer the potential for improved spot detection over currently available software.     

Multidimensional liquid phase protein separations in conjunction with stable isotope labelling for quantitative proteomics

A method for quantitative protein profiling has been developed utilising multidimensional liquid phase protein separations in conjunction with stable isotope labelling. This approach combines the advantages of high throughput, automated, reproducible protein separations with accurate protein quantitation performed in the mass spectrometer. Escherichia coli cells were grown in the presence and absence of the DNA methylation inhibitor 5-Azacytidine on 14N and 15N enriched media. Protein separations were performed using ion exchange chromatography in the first dimension and RP capillary chromatography in the second dimension. UV absorbance measurements were used for the initial semiquantitative identification of differentially expressed proteins. Selected peaks from the mixed 15N/14N lysates were used for the accurate quantitation performed in the mass spectrometer using the ratios of the stable isotopes. Using this approach, a number of differentially expressed proteins have been identified. Moreover, this approach overcomes a number of caveats associated with multidimensional liquid phase protein separations, including the presence of multiple proteins present in a single chromatographic peak.     

A multifaceted analysis of viperid snake venoms by two-dimensional gel electrophoresis: an approach to understanding venom proteomics

The complexity of Viperid venoms has long been appreciated by investigators in the fields of toxinology and medicine. However, it is only recently that the depth of that complexity has become somewhat quantitatively and qualitatively appreciated. With the resurgence of two-dimensional gel electrophoresis (2-DE) and the advances in mass spectrometry virtually all venom components can be visualized and identified given sufficient effort and resources. Here we present the use of 2-DE for examining venom complexity as well as demonstrating interesting approaches to selectively delineate subpopulations of venom proteins based on particular characteristics of the proteins such as antibody cross-reactivity or enzymatic activities. 2-DE comparisons between venoms from different species of the same genus (Bothrops) of snake clearly demonstrated both the similarity as well as the apparent diversity among these venoms. Using liquid chromatography/tandem mass spectrometry we were able to identify regions of the two-dimensional gels from each venom in which certain classes of proteins were found. 2-DE was also used to compare venoms from Crotalus atrox and Bothrops jararaca. For these venoms a variety of staining/detection protocols was utilized to compare and contrast the venoms. Specifically, we used various stains to visualize subpopulations of the venom proteomes of these snakes, including Coomassie, Silver, Sypro Ruby and Pro-Q-Emerald. Using specific antibodies in Western blot analyses of 2-DE of the venoms we have examined subpopulations of proteins in these venoms including the serine proteinase proteome, the metalloproteinase proteome, and the phospholipases A2 proteome. A functional assessment of the gelatinolytic activity of these venoms was also performed by zymography. These approaches have given rise to a more thorough understanding of venom complexity and the toxins comprising these venoms and provide insights to investigators who wish to focus on these venom subpopulations of proteins in future studies.     

Interactome and interface protocol (2IP): a novel strategy for high sensitivity topology mapping of protein complexes

A few well-characterized protein assemblies aside, little is known about the topology and interfaces of multiconstituent protein complexes. Here we report on a novel indirect strategy for low-resolution topology mapping of protein complexes. Following crosslinking, purified protein complexes are subjected to chemical cleavage with cyanogen bromide (CNBr) and the resulting fragments are resolved by 2-D electrophoresis. The side-by-side comparison of a thus generated and a 2-D CNBr fragment map obtained from uncrosslinked material reveals candidate gel spots harboring crosslinked CNBr fragments. In-gel trypsinization and MALDI MS analysis of these informative spots identify the underlying crosslinked CNBr fragments based on unmodified tryptic peptides. Matching the cumulative theoretical molecular mass and predicted pI of these crosslinked CNBr fragments with original gel spot coordinates is required for confident crosslink assignment. The above strategy was successfully validated with the Escherichia coli RNA polymerase (RNAP) core complex and subsequently applied to query the quaternary structure of components of the yeast Skp1-Cdc53/Cullin-F box (SCF) ubiquitin ligase complex. This protocol requires low picomole sample quantities, can be applied to multisubunit protein complexes, and does not rely on specialized data mining software.     

Analysis of the synaptic vesicle proteome using three gel-based protein separation techniques

Synaptic vesicles are key organelles in neurotransmission. Their functions are governed by a unique set of integral and peripherally associated proteins. To obtain a complete protein inventory, we immunoisolated synaptic vesicles from rat brain to high purity and performed a gel-based analysis of the synaptic vesicle proteome. Since the high hydrophobicity of integral membrane proteins hampers their resolution by gel electrophoretic techniques, we applied in parallel three different gel electrophoretic methods for protein separation prior to MS. Synaptic vesicle proteins were subjected to either 1-D SDS-PAGE along with nano-LC ESI-MS/MS or to the 2-D gel electrophoretic techniques benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE, and double SDS (dSDS)-PAGE in combination with MALDI-TOF-MS. We demonstrate that the combination of all three methods provides a comprehensive survey of the proteinaceous inventory of the synaptic vesicle membrane compartment. The identified synaptic vesicle proteins include transporters, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), synapsins, rab and rab-interacting proteins, additional guanine nucleotide triphosphate (GTP) binding proteins, cytoskeletal proteins, and proteins modulating synaptic vesicle exo- and endocytosis. In addition, we identified novel proteins of unknown function. Our results demonstrate that the parallel application of three different gel-based approaches in combination with mass spectrometry permits a comprehensive analysis of the synaptic vesicle proteome that is considerably more complex than previously anticipated.     

Outer membrane vesicles of the VA-MENGOC-BC vaccine against serogroup B of Neisseria meningitidis: Analysis of protein components by two-dimensional gel electrophoresis and mass spectrometry

Neisseria meningitidis is a Gram-negative bacterium responsible for significant mortality worldwide. While effective polysaccharides-based vaccines exist against serogroups A, C, W135, and Y, no similar vaccine is suitable for children under 4 years against disease caused by serogroup B strains. Therefore, major vaccine efforts against this serogroup are based on outer membrane vesicles (OMVs), containing major outer membrane proteins. The OMV-based vaccine produced by the Finlay Institute in Cuba (VA-MENGOC-BC) contributed to the rapid decline of the epidemic in this Caribbean island. While the content of major proteins in this vaccine has been discussed, no detailed work of an outer membrane proteomic map of this, or any other, commercially available OMV-derived product has been published so far. Since OMVs exhibit a large bias toward a few major proteins and usually contain a high content of lipids, establishing the adequate conditions for high resolution, 2-DE of this kind of preparation was definitely a technical challenge. In this work, 2-DE and MS have been used to generate a proteomic map of this product, detailing the presence of 31 different proteins, and it allows the identification of new putative protective protein components it contains.     

Proteomic analysis of protein profiles during early development of the zebrafish, Danio rerio

In the present study, profiles of protein expression were examined during early development of zebrafish, an increasingly popular experimental model in vertebrate development and human diseases. By 2-DE, an initial increase in protein spots from 6 h post-fertilization (hpf) to 8-10 hpf was observed. There was no dramatic change in protein profiles up to 18 hpf, but significant changes occurred in subsequent stages. Interestingly, 49% of the proteins detected at 6 hpf remained detectable by 1 week of age. To map the protein expression patterns in 2-D gels, MALDI-TOF/TOF MS was employed to identify selected protein spots from early embryos. 108 protein spots were found to match known proteins and they were derived from 55 distinct genes. Interestingly, 11 (20%) of them produced multiple protein isoforms or distinct cleavage products. Although deyolked embryos were used in the analysis, a large number of vitellogenin derivatives remained prominently present in the embryos. Other than these, most of the identified proteins are cytosolic, cytoskeletal and nuclear proteins, which are involved in diversified functions such as metabolism, cytoskeleton, translation, protein degradation, etc. Some of the proteins with interesting temporal expression profiles during development are further discussed.     

Potential protein markers for nutritional health effects on colorectal cancer in the mouse as revealed by proteomics analysis

It is suggested that colorectal cancer might be prevented by changes in diet, and vegetable consumption has been demonstrated to have a protective effect. Until now, little is known about the effects of vegetable consumption at the proteome level. Therefore, the effect of increased vegetable intake on the protein expression in the colonic mucosa of healthy mice was studied. Aim was to identify the proteins that are differentially expressed by increased vegetable consumption and to discriminate their possible role in the protection against colorectal cancer. Mice were fed four different vegetable diets, which was followed by analysis of total cellular protein from colonic mucosal cells by a combination of 2-DE and MS. We found 30 proteins that were differentially expressed in one or more diets as compared to the control diet. Six could be identified by MALDI-TOF MS: myosin regulatory light chain 2, carbonic anhydrase I, high-mobility group protein 1, pancreatitis-associated protein 3, glyceraldehyde-3-phosphate dehydrogenase and ATP synthase oligomycin sensitivity conferral protein. Alterations in the levels of these proteins agree with a role in the protection against colon cancer. We conclude that these proteins are suitable markers for the health effect of food on cancer. The observed altered protein levels therefore provide support for the protective effects of vegetables against colorectal cancer.     

A quantitative investigation into the losses of proteins at different stages of a two-dimensional gel electrophoresis procedure

We report the results of a systematic investigation to quantify the losses of protein during a well-established two-dimensional polyacrylamide gel electrophoresis (2-DE) procedure. Radioactively labelled proteins ([(14)C]bovine serum albumin and a homogenate prepared from the liver of a rat that had been injected with [(35)S]methionine) were used, and recovery was quantified by digesting pieces of gel in H(2)O(2) and subjecting the digests to liquid scintillation counting. When samples were loaded onto the first dimension immobilised pH gradient strips by in-gel rehydration, recovery of protein from the strips was 44-80% of the amount of protein loaded, depending on the amount of protein in the sample. Most of the unrecovered protein appeared to have adhered to the reswelling tray. Losses during isoelectric focusing (IEF) were much smaller (7-14%), although approximately 2% of the protein appeared to migrate from sample strips to adjacent blank strips in the focussing apparatus. A further 17-24% of the proteins were lost into the buffers during equilibration prior to running in the second dimension. Losses during the second dimension run and subsequent staining with SYPRO Ruby amounted to less than 10%. The overall loss during 2-DE was reduced by approximately 25% when proteins were loaded onto the IEF strips using sample cups instead of by in-gel rehydration. These extensive and variable losses during the 2-DE procedure mean that spot intensities on 2-DE gels cannot be used to derive reliable, quantitative information on the amounts of proteins present in the original sample.     

Identification of breast cancer metastasis-associated proteins in an isogenic tumor metastasis model using two-dimensional gel electrophoresis and liquid chromatography-ion trap-mass spectrometry

To better understand the molecular mechanisms underlying breast cancer metastasis and search for potential markers for metastatic progression, we have developed a highly metastatic variant of human MDA-MB-435 breast cancer cell line through in vivo stepwise selection of pulmonary metastatic cells caused by parental MDA-MB-435 cells in the athymic mice. Comparative proteomic analysis using 2-DE and LC-IT-MS revealed that 102 protein spots were reproducibly altered more than three-fold between the selected variant and its parental counterpart. Eleven differentially expressed protein spots were identified with high confidence using SEQUEST with uninterpreted tandem mass raw data. Cathepsin D precursor, peroxiredoxin 6 (PDX6), heat shock protein 27 (HSP27), HSP60, tropomyosin 1 (TPM1), TPM2, TPM3, TPM4, 14-3-3 protein epsilon, and tumor protein D54 were up-regulated in the highly metastatic variant, whereas alpha B-crystalline (CRAB) was only detected in its parental counterpart. Differential expression was confirmed for four proteins including PDX6, CRAB, TPM4, and HSP60 by real-time quantitative PCR and Western blotting analysis in our model. Immunohistochemical analysis in 80 breast cancer donors demonstrated a significant association of TPM4 (p = 0.002), HSP60 (p = 0.001), PDX6 (p = 0.002) but not CRAB (p = 0.113) staining with the presence of lymph node metastasis. In addition, TPM4 staining was also associated with clinical stage (p = 0.000), but no significant association was found between TPM4, PDX6, CRAB, and HSP60 expression and tumor size, hormone receptor, and HER-2 status (p > 0.05). The functional implication of these identified proteins was also discussed. These proteomic data are valuable and informative for understanding breast cancer metastasis and searching for potential markers for metastatic progression.