Species differentiation of a diverse suite of Bacillus spores by mass spectrometry-based protein profiling

In this study, we demonstrate the versatility of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOFMS) protein profiling for the species differentiation of a diverse suite of Bacillus spores. MALDI-TOFMS protein profiles of 11 different strains of Bacillus spores, encompassing nine different species, were evaluated. Bacillus species selected for MALDI-TOFMS analysis represented the spore-forming bacterial diversity of typical class 100K clean room spacecraft assembly facilities. A one-step sample treatment and MALDI-TOFMS preparation were used to minimize the sample preparation time. A library of MALDI-TOFMS spectra was created from these nine Bacillus species, the most diverse protein profiling study of the genus reported to date. Linear correlation analysis was used to successfully differentiate the MALDI-TOFMS protein profiles from all strains evaluated in this study. The MALDI-TOFMS protein profiles were compared with 16S rDNA sequences for their bacterial systematics and molecular phylogenetic affiliations. The MALDI-TOFMS profiles were found to be complementary to the 16S rDNA analysis. Proteomic studies of Bacillus subtilis 168 were pursued to identify proteins represented by the biomarker peaks in the MALDI-TOFMS spectrum. Four small, acid-soluble proteins (A, B, C, and D), one DNA binding protein, hypothetical protein ymf J, and four proteins associated with the spore coat and spore coat formation (coat JB, coat F, coat T, and spoIVA) were identified. The ability to visualize higher-molecular-mass coat proteins (10 to 25 kDa) as well as smaller proteins (<10 kDa) with MALDI-TOFMS profiling is critical for the complete and effective species differentiation of the Bacillus genus.     

Chemical probes and tandem mass spectrometry: a strategy for the quantitative analysis of proteomes and subproteomes

Quantitative proteome profiling using mass spectrometry and stable isotope dilution is being widely applied for the functional analysis of biological systems and for the detection of clinical, diagnostic or prognostic marker proteins. Because of the enormous complexity of proteomes, their comprehensive analysis is unlikely to be routinely achieved in the near future. However, in recent years, significant progress has been achieved focusing quantitative proteomic analyses on specific protein classes or subproteomes that are rich in biologically or clinically important information. Such projects typically combine the use of chemical probes that are specific for a targeted group of proteins and may contain stable isotope signatures for accurate quantification with automated tandem mass spectrometry and bioinformatics tools for data analysis. In this review, we summarize technical and conceptual advances in quantitative subproteome profiling based on tandem mass spectrometry and chemical probes.     

MALDI-TOFMS compared with other polyphasic taxonomy approaches for the identification and classification of Bacillus pumilus spores

To verify the efficacy of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) protein profiling for identifying and differentiating bacterial species, several strains of Bacillus pumilus were examined in a thorough taxonomic study incorporating a polyphasic approach. Sixteen isolates of putative B. pumilus isolated from spacecraft assembly facilities, the Mars Odyssey spacecraft, and the International Space Station, were characterized for their biochemical and molecular profiles using the Biolog system, DNA techniques, and MALDI-TOFMS protein profiling. MALDI-TOFMS protein profiling was more accurate than Biolog metabolic profiling, more discriminating than 16S rDNA sequence analysis, and complemented the results of gyrB sequence analysis and DNA-DNA hybridization for the identification of the B. pumilus spores. This is the first report whereby MALDI-TOFMS generated protein profiles from a set of microbes is compared directly with DNA-DNA hybridization yielding a positive correlation. Unique, cluster-specific biomarker peaks have been identified in the spores of the B. pumilus examined in this study. MALDI-TOFMS protein profiling is a rapid and simple analysis and has been demonstrated as a useful taxonomic tool for differentiating spores of the genus Bacillus. For practical purposes, it would be ideal (and necessary) to have a publicly available, standardized MALDI profile database, to facilitate the use of the technique as a diagnostic method to differentiate bacterial species.     

Development of a matrix-assisted laser desorption/ionization-mass spectrometry screening test to evidence reversible and irreversible inhibitors of CDC25 phosphatases

The cell division cycle 25 phosphatases (CDC25s) are key regulators of the physiological cell cycle progression. Their overexpression has been reported in a significant number of cancers, and their inhibition appears to be an interesting strategy for treatments. We propose here a rapid screening test allowing the detection of reversible and irreversible CDC25A and -C inhibitors. The test is based on the incubation of the candidate molecules with the human CDC25 proteins followed by an ultrafiltration step. The retentate is then directly analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOFMS) to detect reversible inhibitors or submitted to peptide mass fingerprint (PMF) analysis to reveal irreversible inhibitors covalently bound to the protein active site. After its validation, the protocol is applied to the detection of a novel candidate inhibitor of CDC25s named SV37. The screening procedure, as well as the preliminary biological results, demonstrates that this compound behaves as a reversible inhibitor.     

A novel antibody microarray format using non-covalent antibody immobilization with chemiluminescent detection

To date, protein and antibody microarrays have been used in reverse-phase and sandwich-based methods in order to detect known proteins such as biomarkers in samples. Our group developed "libraries" of antibodies against unknown proteins, referred to as mKIAA proteins, and we attempted to discover candidate novel biomarkers by protein expression profiling.To profile mKIAA protein expression using these antibodies, we established an antibody microarray system using chemiluminescent detection. A number of techniques for protein-antibody microarrays have been reported; however, no entirely suitable protocol for crude protein samples has been established. To address this issue, we immobilized purified antibodies on hydrophilic surface polymer slides (Maxisorp, Nunc). Although our system is based on the direct labeling of crude protein samples, we achieved sufficient sensitivity (detection limit: 50 pg mL(-1)) and low backgrounds. This sensitivity is on a level with the sandwich immunoassay-based antibody array system. Using our protocol, we developed an antibody microarray spotted with 960 anti-mKIAA antibodies (total: 3888 spots for quadruplicate assessments), and we carried out protein expression profiling of mKIAA proteins. In this study, we generated an expression profile of 960 mKIAA proteins and compared the present results with those obtained via cDNA microarray.     

Combined experimental and statistical strategy for mass spectrometry based serum protein profiling for diagnosis of breast cancer: a case-control study

Serum protein profiling by mass spectrometry is a promising method for early detection of cancer. We have implemented a combined strategy based on matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) and statistical data analysis for serum protein profiling and applied it in a well-described breast cancer case-control study. A rigorous sample collection protocol ensured high quality specimen and reduced bias from preanalytical factors. Preoperative serum samples obtained from 48 breast cancer patients and 28 controls were used to generate MALDI MS protein profiles. A total of nine mass spectrometric protein profiles were obtained for each serum sample. A total of 533 common peaks were defined and represented a 'reference protein profile'. Among these 533 common peaks, we identified 72 peaks exhibiting statistically significant intensity differences ( p < 0.01) between cases and controls. A diagnostic rule based on these 72 mass values was constructed and exhibited a cross-validated sensitivity and specificity of approximately 85% for the detection of breast cancer. With this method, it was possible to distinguish early stage cancers from controls without major loss of sensitivity and specificity. We conclude that optimized serum sample handling and mass spectrometry data acquisition strategies in combination with statistical analysis provide a viable platform for serum protein profiling in cancer diagnosis.     

Technical evaluation of MALDI-TOF mass spectrometry for quantitative proteomic profiling matrix formulation and application

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has been recently used to identify disease markers by directly profiling and quantifying the peptide/proteins in biological samples under different physiological or experimental conditions. The information of reproducibility of such quantitative profiling method has not been available. It is important to evaluate and reduce error from technical variation. In this study, an unbiased signal acquisition strategy was used to evaluate the effects of three sample-matrix spotting methods and two matrix chemicals, α-cyano-4-hydroxycinnamic acid (CHCA) and sinapinic acid, on the reproducibility of the peptide/protein signal intensities. The sandwich spotting method using 0.1% nitrocellulose coating film and CHCA gave the best quantitative results for the standard peptides and proteins with mass<66.5 kDa. The normalized signal intensities of the standard peptides and proteins were directly proportional to their concentrations with intra-assay (within-day) coefficient of variations (CVs) ranging from 6.5% to 17%. When analyzing serum peptides <6000 m/z, the interassay (between-days) CVs of all the evaluated peptide peaks were <15%. These data indicate that with the right MS analysis conditions, MALDI-TOF MS appears to be a feasible tool for directly profiling and quantifying the peptide/ proteins in biological samples.     

Peptide mapping of proteins in cerebrospinal fluid utilizing a rapid preparative two-dimensional electrophoretic procedure and matrix-assisted laser desorption/ionization mass spectrometry

A quick two-step procedure involving liquid phase isoelectric focusing in the Rotofor cell in combination with electroelution in the Mini whole cell gel eluter has been used for purification of proteins from human cerebrospinal fluid (CSF). Fractions, each highly enriched in a single protein band and virtually free of other proteins, were selected for characterization by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOFMS). Six CSF proteins, transferrin, alpha1-acid-glycoprotein, Zn-alpha2-glycoprotein, apolipoprotein A1, apolipoprotein E and beta-trace were identified by MALDI-TOFMS analysis of the tryptic digests. These results demonstrate that the combination of liquid phase IEF and electroelution is a rapid preparative two-dimensional separation which can provide single proteins of high purity, in yields sufficient for characterization by MALDI-TOFMS. Characterization of such brain-specific proteins in CSF will be useful in the investigation of the pathophysiology of different brain disorders.     

Detergent addition to tryptic digests and ion mobility separation prior to MS/MS improves peptide yield and protein identification for in situ proteomic investigation of frozen and formalin‐fixed paraffin‐embedded adenocarcinoma tissue sections

The identification of proteins involved in tumour progression or which permit enhanced or novel therapeutic targeting is essential for cancer research. Direct MALDI analysis of tissue sections is rapidly demonstrating its potential for protein imaging and profiling in the investigation of a range of disease states including cancer. MALDI‐mass spectrometry imaging (MALDI‐MSI) has been used here for direct visualisation and in situ characterisation of proteins in breast tumour tissue section samples. Frozen MCF7 breast tumour xenograft and human formalin‐fixed paraffin‐embedded breast cancer tissue sections were used. An improved protocol for on‐tissue trypsin digestion is described incorporating the use of a detergent, which increases the yield of tryptic peptides for both fresh frozen and formalin‐fixed paraffin‐embedded tumour tissue sections. A novel approach combining MALDI‐MSI and ion mobility separation MALDI‐tandem mass spectrometry imaging for improving the detection of low‐abundance proteins that are difficult to detect by direct MALDI‐MSI analysis is described. In situ protein identification was carried out directly from the tissue section by MALDI‐MSI. Numerous protein signals were detected and some proteins including histone H3, H4 and Grp75 that were abundant in the tumour region were identified.     

Mass spectrometric analysis of protein mixtures at low levels using cleavable 13C-isotope-coded affinity tag and multidimensional chromatography

In order to identify and compare the protein content of very low quantity samples of high complexity, a protocol has been established that combines the differential profiling strength of a new cleavable 13C isotope-coded affinity tag (cICAT) reagent with the high sequence coverage provided by multidimensional liquid chromatography and two modes of tandem mass spectrometry. Major objectives during protocol optimization were to minimize sample losses and establish a robust procedure that employs volatile buffer systems that are highly compatible with mass spectrometry. Cleavable ICAT-labeled tryptic peptides were separated from nonlabeled peptides by avidin affinity chromatography. Subsequently, peptide samples were analyzed by nanoflow liquid chromatography electrospray ionization tandem mass spectrometry and liquid chromatography matrix-assisted laser desorption/ionization tandem mass spectrometry. The use of two ionization/instrumental configurations led to complementary peptide identifications that increased the confidence of protein assignments. Examples that illustrate the power of this strategy are taken from two different projects: i) immunoaffinity purified complexes containing the prion protein from the murine brain, and ii) human tracheal epithelium gland secretions. In these studies, a large number of novel proteins were identified using stringent match criteria, in addition to many that had been identified in previous experiments. In the latter case, the ICAT method produced significant new information on changes that occur in protein expression levels in a patient suffering from cystic fibrosis.     

Global urinary metabolic profiling procedures using gas chromatography-mass spectrometry

The role of urinary metabolic profiling in systems biology research is expanding. This is because of the use of this technology for clinical diagnostic and mechanistic studies and for the development of new personalized health care and molecular epidemiology (population) studies. The methodologies commonly used for metabolic profiling are NMR spectroscopy, liquid chromatography mass spectrometry (LC/MS) and gas chromatography-mass spectrometry (GC/MS). In this protocol, we describe urine collection and storage, GC/MS and data preprocessing methods, chemometric data analysis and urinary marker metabolite identification. Results obtained using GC/MS are complementary to NMR and LC/MS. Sample preparation for GC/MS analysis involves the depletion of urea via treatment with urease, protein precipitation with methanol, and trimethylsilyl derivatization. The protocol described here facilitates the metabolic profiling of ～400-600 metabolites in 120 urine samples per week.     

Gas chromatography mass spectrometry-based metabolite profiling in plants

The concept of metabolite profiling has been around for decades, but technical innovations are now enabling it to be carried out on a large scale with respect to the number of both metabolites measured and experiments carried out. Here we provide a detailed protocol for gas chromatography mass spectrometry (GC-MS)-based metabolite profiling that offers a good balance of sensitivity and reliability, being considerably more sensitive than NMR and more robust than liquid chromatography-linked mass spectrometry. We summarize all steps from collecting plant material and sample handling to derivatization procedures, instrumentation settings and evaluating the resultant chromatograms. We also define the contribution of GC-MS-based metabolite profiling to the fields of diagnostics, gene annotation and systems biology. Using the protocol described here facilitates routine determination of the relative levels of 300-500 analytes of polar and nonpolar extracts in approximately 400 experimental samples per week per machine.     

定量蛋白质组学中的同位素标记技术

定量蛋白质组学的目的是对复杂的混合体系中所有的蛋白质进行鉴定,并对蛋白质的量及量的变化进行准确的测定,是当前系统生物科学研究的重要内容。近年来,由于质谱技术和生物信息学的进步,定量蛋白质组学在分析蛋白质组或亚蛋白质组方面已取得了令人瞩目的成就,但其最显著的成就应该归功于稳定同位素标记技术的应用。该技术使用针对某一类蛋白具有特异性的化学探针来标记目的蛋白质或肽段,同时化学探针要求含有用以精确定量的稳定同位素信号。在此基础上,实现了对表达的蛋白质差异和翻译后修饰的蛋白质差异进行精确定量分析。综述了在定量蛋白质组学中使用的各种同位素标记技术及其应用。     

Lectin capture strategies combined with mass spectrometry for the discovery of serum glycoprotein biomarkers

The application of mass spectrometry to identify disease biomarkers in clinical fluids like serum using high throughput protein expression profiling continues to evolve as technology development, clinical study design, and bioinformatics improve. Previous protein expression profiling studies have offered needed insight into issues of technical reproducibility, instrument calibration, sample preparation, study design, and supervised bioinformatic data analysis. In this overview, new strategies to increase the utility of protein expression profiling for clinical biomarker assay development are discussed with an emphasis on utilizing differential lectin-based glycoprotein capture and targeted immunoassays. The carbohydrate binding specificities of different lectins offer a biological affinity approach that complements existing mass spectrometer capabilities and retains automated throughput options. Specific examples using serum samples from prostate cancer and hepatocellular carcinoma subjects are provided along with suggested experimental strategies for integration of lectin-based methods into clinical fluid expression profiling strategies. Our example workflow incorporates the necessity of early validation in biomarker discovery using an immunoaffinity-based targeted analytical approach that integrates well with upstream discovery technologies.     

Multidimensional protein profiling technology and its application to human plasma proteome

In clinical and diagnostic proteomics, it is essential to develop a comprehensive and robust system for proteome analysis. Although multidimensional liquid chromatography/tandem mass spectrometry (LC/MS/MS) systems have been recently developed as powerful tools especially for identification of protein complexes, these systems still some drawbacks in their application to clinical research that requires an analysis of a large number of human samples. Therefore, in this study, we have constructed a technically simple and high throughput protein profiling system comprising a two-dimensional (2D)-LC/MS/MS system which integrates both a strong cation exchange (SCX) chromatography and a microLC/MS/MS system with micro-flowing reversed-phase chromatography. Using the microLC/MS/MS system as the second dimensional chromatography, SCX separation has been optimized as an off-line first dimensional peptide fractionation. To evaluate the performance of the constructed 2D-LC/MS/MS system, the results of detection and identification of proteins were compared using digests mixtures of 6 authentic proteins with those obtained using one-dimensional microLC/MS/MS system. The number of peptide fragments detected and the coverage of protein sequence were found to be more than double through the use of our newly built 2D-LC/MS/MS system. Furthermore, this multidimensional protein profiling system has been applied to plasma proteome in order to examine its feasibility for clinical proteomics. The experimental results revealed the identification of 174 proteins from one serum sample depleted HSA and IgG which corresponds to only 1 microL of plasma, and the total analysis run time was less than half a day, indicating a fairly high possibility of practicing clinical proteomics in a high throughput manner.     

Multiplexed complexome profiling using tandem mass tags

Complexome profiling is a rapidly spreading, powerful technique to gain insight into the nature of protein complexes. It identifies and quantifies protein complexes separated into multiple fractions of increasing molecular mass using mass spectrometry-based, label-free bottom-up proteomics. Complexome profiling enables a sophisticated and thorough characterization of the composition, molecular mass, assembly, and interactions of protein complexes. However, in practice, its application is limited by the large number of samples it generates and the related time of mass spectrometry analyses. Here, we report an improved process workflow that implements tandem mass tags for multiplexing complexome profiling. This workflow substantially reduces the number of samples and measuring time without compromising protein identification or quantification reliability. In profiles from mitochondrial fractions of cells recovering from chloramphenicol treatment, tandem mass tags-multiplexed complexome profiling exhibited migration patterns of mature ATP synthase (complex V) and assembly intermediates that were consistent in composition and abundance with profiles obtained by the label-free approach. Reporter ion quantifications of proteins and complexes unaffected by the chloramphenicol treatment presented less variation in comparison to the label-free method. Incorporation of tandem mass tags enabled an efficient and robust complexome profiling analysis and may foster broader application for protein complex profiling in biomedical research and diagnostics.     

Tissue-specific microdissection coupled with ProteinChip array technologies: applications in cancer research

Analysis of whole genomes to monitor specific changes in gene activation or changes in gene copy number due to perturbation has recently become possible using DNA chip technologies. It is now becoming apparent, however, that knowing the genetic sequence encoding a protein is not sufficient to predict the size or biological nature of a protein. This can be particularly important in cancer research where posttranslational modifications of a protein can specifically lead to the disease. To address this area, several proteomic tools have been developed. Currently the most widely used proteomics tool is two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), which can display protein expression patterns to a high degree of resolution. However, 2D-PAGE can be time consuming; the analysis is complicated and, compared with DNA techniques, is not very sensitive. Although some of these problems can be alleviated by using high-quality homogeneous samples, such as those generated using microdissection techniques, the quantity of sample is often limited and may take several days to generate sufficient material for a single 2D-PAGE analysis. As an alternative to 2D-PAGE, a preliminary study using a new technique was used to generate protein expression patterns from either whole tissue extracts or microdissected material. Surface-enhanced laser desorption and ionization allows the retention of proteins on a solid-phase chromatographic surface or ProteinChip Array with direct detection of retained proteins by time-of-flight mass spectrometry. Using this system, we analyzed tumor and normal tissue from head and neck cancer and microdissected melanoma to determine differentially expressed proteins. In particular, comparisons of the protein expression patterns from microdissected normal and tumor tissues indicated several differences, highlighting the importance of extremely defined tissue lysates for protein profiling.     

Proteome analysis of low-abundance proteins using multidimensional chromatography and isotope-coded affinity tags

The effectiveness of proteome-wide protein identification and quantitative expression profiling is dependent on the ability of the analytical methodologies employed to routinely obtain information on low-abundance proteins, as these are frequently of great biological importance. Two-dimensional gel electrophoresis, the traditional method for proteome analysis, has proven to be biased toward highly expressed proteins. Recently, two-dimensional chromatography of the complex peptide mixtures generated by the digestion of unseparated protein samples has been introduced for the identification of their components, and isotope-coded affinity tags (ICAT) have been introduced to allow for accurate quantification of the components of protein mixtures by mass spectrometry. Here, we demonstrate that the combination of isotope coded affinity protein tags and multidimensional chromatography/mass spectrometry of tryptic peptide mixtures is capable of detecting and quantifying proteins of low abundance in complex samples.     

Overcoming the dynamic range problem in mass spectrometry-based shotgun proteomics

Protein profiling using mass spectrometry technology has emerged as a powerful method for analyzing large-scale protein-expression patterns in cells and tissues. However, a number of challenges are present in proteomics research, one of the greatest being the high degree of protein complexity and huge dynamic range of proteins expressed in the complex biological mixtures, which exceeds six orders of magnitude in cells and ten orders of magnitude in body fluids. Since many important signaling proteins have low expression levels, methods to detect the low-abundance proteins in a complex sample are required. This review will focus on the fundamental fractionation and mass spectrometry techniques currently used for large-scale shotgun proteomics research.