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The potentials of MS-based subproteomic approaches in medical science: the case of lysosomes and breast cancer.

Because of the great number of women who are diagnosed with breast cancer each year, and though this disease presents the lowest mortality rate among cancers, breast cancer remains a major public health problem. As for any cancer, the tumorigenic and metastatic processes are still hardly understood, and the biochemical markers that allow either a precise monitoring of the disease or the classification of the numerous forms of breast cancer remain too scarce. Therefore, great hopes are put on the development of high-throughput genomic and proteomic technologies. Such comprehensive techniques should help in understanding the processes and in defining steps of the disease by depicting specific genes or protein profiles. Because techniques dedicated to the current proteomic challenges are continuously improving, the probability of the discovery of new potential protein biomarkers is rapidly increasing. In addition, the identification of such markers should be eased by lowering the sample complexity; e.g., by sample fractionation, either according to specific physico-chemical properties of the proteins, or by focusing on definite subcellular compartments. In particular, proteins of the lysosomal compartment have been shown to be prone to alterations in their localization, expression, or post-translational modifications (PTMs) during the cancer process. Some of them, such as the aspartic protease cathepsin D (CatD), have even been proven as participating actively in the disease progression. The present review aims at giving an overview of the implication of the lysosome in breast cancer, and at showing how subproteomics and the constantly refining MS-based proteomic techniques may help in making breast cancer research progress, and thus, hopefully, in improving disease treatment.

Biomarkers, Tumor↗

PubMed2004

ProteoMod: A new tool to quantitate protein post-translational modifications.

Post-translational modifications (PTMs) are known to regulate biological processes by controlling protein function. The effect of a PTM on protein function depends critically on the position and the number of modifications. While there are convenient methods available to qualitatively examine modifications like phosphorylation, glycosylation, acetylation and methylation, methods available for their quantitative assessment are cumbersome. We have developed a new tool that allows quantitation of the number of phosphorylation events in proteins with ease. The "ProteoMod" tool depends on shifts in the isoelectric points of proteins upon post-translational change. The extent of shift exhibited upon phosphorylation is algorithmically converted into the number of phosphorylations conferred. The validity of ProteoMod was confirmed by examining proteins with previously known number of phosphorylations. The list of proteins examined included HSP27, HSP70 and tumor suppressor p53. The approach can also be applied to estimate modifications like acetylation, methylation and sialylation in proteins. We analyzed shifts in isoelectric points due to sialylation events in N-glycoproteins. Using influenza hemagglutinin we show that shifts in isoelectric points correlate with intracellular distribution of this model membrane protein. In addition to extending the application of two dimensional gel electrophoresis to quantitate modifications, our study also highlights its potential use in cell biology.

Algorithms↗

PubMed2004

Quantify this! Report on a round table discussion on quantitative mass spectrometry in proteomics.

Following the success of the first round table in 2001, the Swiss Proteomic Society has organized two additional specific events during its last two meetings: a proteomic application exercise in 2002 and a round table in 2003. Such events have as their main objective to bring together, around a challenging topic in mass spectrometry, two groups of specialists, those who develop and commercialize mass spectrometry equipment and software, and expert MS users for peptidomics and proteomics studies. The first round table (Geneva, 2001) entitled "Challenges in Mass Spectrometry" was supported by brief oral presentations that stressed critical questions in the field of MS development or applications (Stöcklin and Binz, Proteomics 2002, 2, 825-827). Topics such as (i) direct analysis of complex biological samples, (ii) status and perspectives for MS investigations of noncovalent peptide-ligant interactions; (iii) is it more appropriate to have complementary instruments rather than a universal equipment, (iv) standardization and improvement of the MS signals for protein identification, (v) what would be the new generation of equipment and finally (vi) how to keep hardware and software adapted to MS up-to-date and accessible to all. For the SPS'02 meeting (Lausanne, 2002), a full session alternative event "Proteomic Application Exercise" was proposed. Two different samples were prepared and sent to the different participants: 100 micro g of snake venom (a complex mixture of peptides and proteins) and 10-20 micro g of almost pure recombinant polypeptide derived from the shrimp Penaeus vannamei carrying an heterogeneous post-translational modification (PTM). Among the 15 participants that received the samples blind, eight returned results and most of them were asked to present their results emphasizing the strategy, the manpower and the instrumentation used during the congress (Binz et. al., Proteomics 2003, 3, 1562-1566). It appeared that for the snake venom extract, the quality of the results was not particularly dependant on the strategy used, as all approaches allowed Lication of identification of a certain number of protein families. The genus of the snake was identified in most cases, but the species was ambiguous. Surprisingly, the precise identification of the recombinant almost pure polypeptides appeared to be much more complicated than expected as only one group reported the full sequence. Finally the SPS'03 meeting reported here included a round table on the difficult and challenging task of "Quantification by Mass Spectrometry", a discussion sustained by four selected oral presentations on the use of stable isotopes, electrospray ionization versus matrix-assisted laser desorption/ionization approaches to quantify peptides and proteins in biological fluids, the handling of differential two-dimensional liquid chromatography tandem mass spectrometry data resulting from high throughput experiments, and the quantitative analysis of PTMs. During these three events at the SPS meetings, the impressive quality and quantity of exchanges between the developers and providers of mass spectrometry equipment and software, expert users and the audience, were a key element for the success of these fruitful events and will have definitively paved the way for future round tables and challenging exercises at SPS meetings.

Animals↗

PubMed2005

A proteomic method for the analysis of changes in protein concentrations in response to systemic perturbations using metabolic incorporation of stable isotopes and mass spectrometry.

While several techniques exist for assessing quantitative differences among proteomes representing different cell states, methods for assessing how these differences are mediated are largely missing. We present a method that allows one to differentiate between cellular processes, such as protein synthesis, degradation and PTMs which affect protein concentrations. An induced systemic perturbation of a cell culture was coupled to a replacement of the growth medium to one highly enriched in the stable isotope 15N. The relative abundance of the 15N- and 14N-enriched forms of proteins, isolated from cell cultures harvested at time points following the onset of the perturbation, were determined by MS. Alterations in protein synthesis and degradation were quantified by comparing proteins isolated from perturbed and unperturbed cultures, respectively. The method was evaluated by subjecting HeLa cells to heat stress. As expected, a number of known heat shock proteins (Hsp) increased in concentration during heat stress. For Hsp27, increased de novo synthesis accounted for the concentration increase, while for Hsp70, decreased degradation accounted for the increase. A protein that was detected only after prolonged heat stress, vimentin, was not primarily synthesized de novo, but appeared rather as a result of PTM.

Half-Life↗

PubMed2005

Proteomic analysis of organ-specific post-translational lysine-acetylation and -methylation in mice by use of anti-acetyllysine and -methyllysine mouse monoclonal antibodies.

Post-translational lysine-acetylation and -methylation are two major PTMs of lysine residues in proteins. Recently, we established pan-reactive anti-acetyllysine mouse mAbs, which can bind to Nepsilon-acetylated lysine residues in various contexts of amino acid sequences. In the present study, we established pan-reactive anti-methyllysine mouse mAbs comparable to the anti-acetyllysine ones. By using these anti-acetyllysine and -methyllysine antibodies, we found that the pattern of lysine-acetylated and -methylated proteins in mouse organs showed extreme variation from organ to organ. We selected brain and skeletal muscle as model cases to be further analyzed by 2-DE followed by Western blotting. In brain, alpha-tubulin at its basal level was found to be extremely acetylated; and alpha-enolase was shown to be a newly recognized possibly acetylated protein. NF-L protein, Hsc70, alpha-tubulin fragments, beta-actin, and brain-type creatine kinase were identified as putative lysine-methylated proteins in mouse brain. In skeletal muscle, lysine-methylation of alpha-actin and both lysine-acetylation and -methylation of muscle-type creatine kinase were found as novel putative lysine-modified proteins. The approach presented here might be useful to find novel disease markers and/or drug target molecules that would not be noticed by use of the traditional proteomic approach only.

Acetylation↗

PubMed2005

A wide range of protein isoforms in serum and plasma uncovered by a quantitative intact protein analysis system.

We have implemented an orthogonal 3-D intact protein analysis system (IPAS) to quantitatively profile protein differences between human serum and plasma. Reference specimens consisting of pooled Caucasian-American serum, citrate-anticoagulated plasma, and EDTA-anticoagulated plasma were each depleted of six highly abundant proteins, concentrated, and labeled with a different Cy dye (Cy5, Cy3, or Cy2). A mixture consisting of each of the labeled samples was subjected to three dimensions of separation based on charge, hydrophobicity, and molecular mass. Differences in the abundance of proteins between each of the three samples were determined. More than 5000 bands were found to have greater than two-fold difference in intensity between any pair of labeled specimens by quantitative imaging. As expected, some of the differences in band intensities between serum and plasma were attributable to proteins related to coagulation. Interestingly, many proteins were identified in multiple fractions, each exhibiting different pI, hydrophobicity, or molecular mass. This is likely reflective of the expression of different protein isoforms or specific protein cleavage products, as illustrated by complement component 3 precursor and clusterin. IPAS provides a high resolution, high sensitivity, and quantitative approach for the analysis of serum and plasma proteins, and allows assessment of PTMs as a potential source of biomarkers.

Anticoagulants↗

PubMed2005

Proteome analysis of cortical neuronal cultures following cycloheximide, heat stress and MK801 preconditioning.

Studying endogenous neuroprotective mechanisms induced by preconditioning may provide drug leads to reduce ischemic neuronal death. In this study, we used 2-DE to examine protein expression following cycloheximide, heat stress, and MK801 preconditioning in rat cortical neuronal cultures. Of 150 differentially expressed protein spots selected for identification the protein or tentative protein(s) were identified in 84 cases, representing 50 different proteins. Different protein spots representing the same protein or closely related protein(s) occurred for 21 of the identified proteins and are likely to represent PTMs or proteolytic fragments of the protein. Six protein spots (actin, elongation factor 1-alpha 1, peptidyl-prolyl cis-transisomerase A, Cu/Zn superoxide dismutase, stathmin, tropomyosin) were differentially expressed in all three preconditioning treatments. Twenty-seven protein spots were differentially expressed in two preconditioning treatments, while 51 spots were differentially expressed in one treatment. Three proteins heterogeneous nuclear ribonucleoproteins A2/B1, mitochondrial stress-70 protein, and tropomyosin were detected in control neuronal cultures, but not following one or more preconditioning treatments, while a posttranslational modified form of the voltage dependent anion channel 1 was only detected following cycloheximide preconditioning. In summary, this study has revealed multiple protein changes potentially involved in neuroprotective and neurodamaging pathways, which require further characterization.

Animals↗

PubMed2006

Visualisation and analysis of proteomic data from the procyclic form of Trypanosoma brucei.

We have undertaken a large scale study of the proteins expressed in the procyclic form of the parasite Trypanosoma brucei, which causes African sleeping sickness, using 2-DE and MS. The complete data set encompasses over 2000 identifications, of which 770 are distinct proteins. We have discovered that multiple protein isoforms appear to be common in T. brucei, as most proteins have been matched to more than one gel spot. We have developed visualisation software to investigate the differences between isoforms, based on the information from the results of database searches with MS data. We are able to highlight instances where PTMs are the most likely cause of variant forms. In other cases, spots that appear reproducibly across replicates contain fragments of proteins, arising either as experimental artefacts or as part of protein degradation. We are also able to classify clusters of gel spots into different groups based on the pattern of peptides that have been matched from MS data. The entire data set is stored within a relational database system that allows complex queries ( http://www.gla.ac.uk/functionalgenomics). Using specific proteins as examples, we demonstrate how the visualisation software and the database query facilities can be used.

Animals↗

PubMed2006

Development of a "reverse capture" autoantibody microarray for studies of antigen-autoantibody profiling.

Diagnosing cancers based on serum profiling is a particularly attractive concept. However, the technical challenges to analysis of the serum proteome arise from the dynamic range of protein amounts. Cancer sera contain antibodies that react with a unique group of autologous cellular antigens, which affords a dramatic amplification of signal in the form of antibodies relative to the amount of the corresponding antigens. The serum autoantibody repertoire from cancer patients might, therefore, be exploited for antigen-antibody profiling. To date, studies of antigen-antibody reactivity using microarrays have relied on recombinant proteins or synthetic peptides as arrayed features. However, recombinant proteins and/or synthetic peptides may fail to accurately detect autoantibody binding due to the lack of proper PTMs. Here we describe the development and use of a "reverse capture" autoantibody microarray. Our "reverse capture" autoantibody microarray is based on the dual-antibody sandwich immunoassay platform of ELISA, which allows the antigens to be immobilized in their native configuration. As "proof-of-principle", we demonstrate its use for antigen-autoantibody profiling with sera from patients with prostate cancer and benign prostate hyperplasia.

Aged↗

PubMed2006

Human Disease Glycomics/Proteome Initiative Workshop and the 4th HUPO Annual Congress.

At the Human Disease Glycomics/Proteome Initiative workshop held on August 27 on the occasion of the fourth HUPO Congress in Munich, Germany, the working groups reported on pilot studies, performed by 21 laboratories world-wide, to analyze glycan using MS. During the steering committee on July 31 in Osaka, Japan, it was reported that these groups have developed a system using MS to assess the world-wide network on the screening of congenital disorders of glycosylation. The concept of glycobioinformatics was also explained at this meeting. The session on PTMs laid particular emphasis on the importance of functional glycomics using glycosyltransferase genes as well as on the importance of identifying the target protein(s) carrying the sugar chains.

Carbohydrates↗

PubMed2006

Proteomic analysis of fast and slow muscles from normal and kyphoscoliotic mice using protein arrays, 2-DE and MS.

A proteomic strategy based upon the integrated use of SELDI-TOF/MS, 2-DE and MALDI-TOF/MS has been used to identify a panel of fast muscle protein markers: MLC1F, MLC3F, fast troponin C (STNC) and slow muscle markers: MLC1SB and MLC2v. MLC3F, MLC1F and STNC were virtually absent in the physiologically pure slow soleus muscle of kyphoscoliotic mutant mice compared to control BDmice, whereas MLC2v increased threefold. A SELDI-TOF/MS peak at 18,012 Da in spectra from strong anionic exchange protein array fractions of fast vastus muscle was confirmed as STNC by its specific depletion from crude extracts of vastus muscle using an anti-TNC mAb. SELDI-TOF/MS also identified MLC2F phosphorylation in crude muscle extracts after treatment with alkaline phosphatase. High probability protein identifications were achieved by SELDI-TOF/MS PMF based upon the resolution of large peptides formed by partial cleavage and high peptide coverage. When the pI from 2-D gels and molecular weight estimations from SELDI-TOF/MS were entered into the TagIdent algorithm, high probability protein identity predictions were obtained that were confirmed later by PMF. We confirm that SELDI-TOF/MS can be integrated with other proteomics techniques for the efficient analysis of protein expression changes and PTMs associated with physiological changes in skeletal muscle.

Animals↗

PubMed2006

Comprehensive glyco-proteomic analysis of human alpha1-antitrypsin and its charge isoforms.

Human alpha1-antitrypsin (A1PI) is a well-known glycoprotein in human plasma important for the protection of tissues from proteolytic enzymes. The three N-glycosylation sites of A1PI contain diantennary N-glycans but also triantennary and even traces of tetraantennary structures leading to the typical IEF pattern observed for A1PI. Here we present an approach to characterize A1PI isoforms from human plasma and its PTMs by LC-ESI-MS and LC-ESI-MS/MS of peptides obtained by proteolytic digestion. The single cysteine residue of A1PI formed a disulfide bridge with free cysteine. The variability of the number of antennae and hence sialic acids on glycosylation site N107, which even contained minute amounts of tetraantennary structures, emerged as a major cause for the IEF pattern of A1PI. Only negligible amounts of triantennary structures were identified attached to N70, and exclusively diantennary structures were present on site N271 in each of the isoforms analyzed. Exoglycosidase digests revealed alpha2,6-linked neuraminic acids on diantennary N-glycans, and triantennary contained additionally one single alpha2,3-neuraminic acid per N-glycan, which, together with a fucose, formed a sialyl Lewis X determinant on the beta1,4-linked N-acetylglucosamine, as shown by 2-D-HPLC of pyridylaminated asialoglycans. Fucosylation of diantennary structures was marginal and of the core alpha1,6 type.

Carbohydrate Conformation↗

PubMed2006

Analysis of acidic peptides with a matrix-assisted laser desorption/ionization mass spectrometry using positive and negative ion modes with additive monoammonium phosphate.

Acidic PTMs such as phosphorylation and sulfonation of proteins are known to play important roles in many cellular processes including signal transductions and protein-protein interactions. In MS, the acidic modified peptides, that have negative charge, are observable in negative ion mode rather than in positive ion mode. Moreover, addition of ammonium salt into MALDI matrix solution improves the relative intensity of ionization of the phosphorylated peptide to unmodified one. We demonstrate that a combination of the negative ion mode and addition of ammonium salt is more effective in the ionization of the acidic modified peptides. We applied this method to 2-DE separated proteins of Caenorhabditis elegans. As a result, 42 spots were identified as modified proteins, of which 34 proteins were nonoverlapping unique proteins. Furthermore, our study revealed that pI shifts of the DIM-1 and MLC-1 proteins in the 2-DE gel were attributed to the presence of the acidic modifications. The negative ion mode together with the addition of ammonium salt provides us a useful method to detect the phosphorylation and/or sulfonation of protein in a simple manner.

Caenorhabditis elegans Proteins↗

PubMed2006

The use of mass spectrometry for the proteomic analysis of glycosylation.

Of all protein PTMs, glycosylation is by far the most common, and is a target for proteomic research. Glycosylation plays key roles in controlling various cellular processes and the modifications of the glycan structures in diseases highlight the clinical importance of this PTM. Glycosylation analysis remains a difficult task. MS, in combination with modern separation methodologies, is one of the most powerful and versatile techniques for the structural analysis of glycoconjugates. This review describes methodologies based on MS for detailed characterization of glycoconjugates in complex biological samples at the sensitivity required for proteomic work.

Biomarkers↗

PubMed2006

Hypophosphorylation of the architectural chromatin protein DEK in death-receptor-induced apoptosis revealed by the isotope coded protein label proteomic platform.

During apoptosis nuclear morphology changes dramatically due to alterations of chromatin architecture and cleavage of structural nuclear proteins. To characterize early events in apoptotic nuclear dismantling we have performed a proteomic study of apoptotic nuclei. To this end we have combined a cell-free apoptosis system with a proteomic platform based on the differential isotopic labeling of primary amines with N-nicotinoyloxy-succinimide. We exploited the ability of this system to produce nuclei arrested at different stages of apoptosis to analyze proteome alterations which occur prior to or at a low level of caspase activation. We show that the majority of proteins affected at the onset of apoptosis are involved in chromatin architecture and RNA metabolism. Among them is DEK, an architectural chromatin protein which is linked to autoimmune disorders. The proteomic analysis points to the occurrence of multiple PTMs in early apoptotic nuclei. This is confirmed by showing that the level of phosphorylation of DEK is decreased following apoptosis induction. These results suggest the unexpected existence of an early crosstalk between cytoplasm and nucleus during apoptosis. They further establish a previously unrecognized link between DEK and cell death, which will prove useful in the elucidation of the physiological function of this protein.

Animals↗

PubMed2006

Proteomics of ischemia and reperfusion injuries in rabbit myocardium with and without intervention by an oxygen-free radical scavenger.

A brief period of ischemia followed by timely reperfusion may lead to prolonged, yet reversible, contractile dysfunction (myocardial stunning). Damage to the myocardium occurs not only during ischemia, but also during reperfusion, where a massive release of oxygen-free radicals (OFR) occurs. We have previously utilized 2-DE and MS to define 57 protein spot changes during brief ischemia/reperfusion (15 min ischemia, 60 min reperfusion; 15I/60R) injury in a rabbit model (White, M. Y., Cordwell, S. J., McCarron, H. C. K., Prasan, A. M. et al., Proteomics 2005, 5, 1395-1410) and shown that the majority of these occur because of physical and/or chemical PTMs. In this study, we subjected rabbit myocardium to 15I/60R in the presence of the OFR scavenger N-(2-mercaptopropionyl) glycine (MPG). Thirty-seven of 57 protein spots altered during 15I/60R remained at control levels in the presence of MPG (15I/60R + MPG). Changes to contractile proteins, including myosin light chain 2 (MLC-2) and troponin C (TnC), were prevented by the addition of MPG. To further investigate the individual effects of ischemia and reperfusion, we generated 2-DE gels from rabbit myocardium subjected to brief ischemia alone (15I/0R), and observed alterations of 33 protein spots, including 18/20 seen in both 15I/60R-treated and 15I/60R + MPG-treated tissue. The tissue was also subjected to ischemia in the presence of MPG (15I/0R + MPG), and 21 spot changes, representing 14 protein variants, remained altered despite the presence of the OFR scavenger. These ischemia-specific proteins comprised those involved in energy metabolism (lactate dehydrogenase and ATP synthase alpha), redox regulation (NADH ubiquinone oxidoreductase 51 kDa and GST Mu), and stress response (Hsp27 and 70, and deamidated alpha B-crystallin). We conclude that contractile dysfunction associated with myocardial stunning is predominantly caused by OFR damage at the onset of reperfusion, but that OFR-independent damage also occurs during ischemia. These ischemia-specific protein modifications may be indicative of early myocardial injury.

Animals↗

PubMed2006

Rejuvenating rice proteomics: facts, challenges, and visions.

Proteomics is progressing at an unprecedented pace, as can be exemplified by the progress in model organisms such as yeast, bacteria, and mammals. Proteomics research in plants, however, has not progressed at the same pace. Unscrambling of the genome sequences of the dicotyledoneous Arabidopsis thaliana (L.) and monocotyledoneous rice (Oryza sativa L.) plant species, respectively, has made them accessible reference organisms to study plant proteomics. Study of these two reference plants is expected to unravel the mystery of plant biology. Rice, a critically important food crop on the earth, has been termed a "cornerstone" and the "Rosetta stone" for functional genomics of cereal crops. Here, we look at the progress in unraveling rice proteomes and present the facts, challenges, and vision. The text is divided into two major parts: the first part presents the facts and the second part discusses the challenges and vision. The facts include the technology and its use in developing proteomes, which have been critically and constructively reviewed. The challenges and vision deal with the establishment of technologies to exhaustively investigate the protein components of a proteome, to generate high-resolution gel-based reference maps, and to give rice proteomics a functional dimension by studying PTMs and isolation of multiprotein complexes. Finally, we direct a vision on rice proteomics. This is our third review in series on rice proteomics, which aims to stimulate an objective discussion among rice researchers and to understand the necessity and impact of unraveling rice proteomes to their full potential.

Arabidopsis↗

PubMed2006

Chemistry meets proteomics: the use of chemical tagging reactions for MS-based proteomics.

As proteomics matures from a purely descriptive to a function-oriented discipline of the life sciences, there is strong demand for novel methodologies that increase the depth of information that can be obtained from proteomic studies. MS has long played a central role for protein identification and characterization, often in combination with dedicated chemical modification reactions. Today, chemistry is helping to advance the field of proteomics in numerous ways. In this review, we focus on those methodologies that have a significant impact for the large-scale study of proteins and peptides. This includes approaches that allow the introduction of affinity tags for the enrichment of subclasses of peptides or proteins and strategies for in vitro stable isotope labeling for quantification purposes, among others. Particular attention is given to the study of PTMs where recent advancements have been promising, but many interesting targets are not yet being addressed.

Animals↗