IQuant: An automated pipeline for quantitative proteomics based upon isobaric tags

Quantitative proteomics technology based on isobaric tags is playing an important role in proteomic investigation. In this paper, we present an automated software, named IQuant, which integrates a postprocessing tool of protein identification and advanced statistical algorithms to process the MS/MS signals generated from the peptides labeled by isobaric tags and aims at proteomics quantification. The software of IQuant, which is freely downloaded at http://sourceforge.net/projects/iquant/ , can run from a graphical user interface and a command‐line interface, and can work on both Windows and Linux systems.     

Proteomics without polyacrylamide: qualitative and quantitative uses of tandem mass spectrometry in proteome analysis

Proteomics can be thought of as an attempt to understand the information encoded in genomic sequences from the perspective of proteins; i.e. the structure, function and regulation of biological processes at the protein level. In practice it stands in stark contrast to the hypothesis-driven serial approach practiced in the last century that was so successful for protein chemists and is built on the basic understanding of protein physicochemical properties developed during that era. Proteomics attempts to study biological processes comprehensively or globally by systematic parallel analysis of proteins expressed in a cell. While there are many analytical techniques in use and under development in proteomics, mass spectrometry is currently one of the field's most important discovery-based tools. This article will review some of the current approaches for qualitative and quantitative uses of tandem mass spectrometry in the field of proteomics specifically avoiding a discussion of the use of gel electrophoresis prior to mass spectrometry. Electronic Publication     

Advances in qualitative and quantitative plant membrane proteomics

The membrane proteome consists of integral and membrane-associated proteins that are involved in various physiological and biochemical functions critical for cellular function. It is also dynamic in nature, where many proteins are only expressed during certain developmental stages or in response to environmental stress. These proteins can undergo post-translational modifications in response to these different conditions, allowing them to transiently associate with the membrane or other membrane proteins. Along with their increased size, hydrophobicity, and the additional organelle and cellular features of plant cells relative to mammalian systems, the characterization of the plant membrane proteome presents unique challenges for effective qualitative and quantitative analysis using mass spectrometry (MS) analysis. Here, we present the latest advancements developed for the isolation and fractionation of plant organelles and their membrane components amenable to MS analysis. Separations of membrane proteins from these enriched preparations that have proven effective are discussed for both gel- and liquid chromatography-based MS analysis. In this context, quantitative membrane proteomic analyses using both isotope-coded and label-free approaches are presented and reveal the potential to establish a wider-biological interpretation of the function of plant membrane proteins that will ultimately lead to a more comprehensive understanding of plant physiology and their response mechanisms.     

A proteomics assay to detect eight CBRN‐relevant toxins in food

A proteomics assay was set up to analyze food substrates for eight toxins of the CBRN (chemical, biological, radiological and nuclear) threat, namely ricin, Clostridium perfringens epsilon toxin (ETX), Staphylococcus aureus enterotoxins (SEA, SEB and SED), shigatoxins from Shigella dysenteriae and entero‐hemorragic Escherichia coli strains (STX1 and STX2) and Campylobacter jejuni cytolethal distending toxin (CDT). The assay developed was based on an antibody‐free sample preparation followed by bottom‐up LC‐MS/MS analysis operated in targeted mode. Highly specific detection and absolute quantification were obtained using isotopically labeled proteins (PSAQ standards) spiked into the food matrix. The sensitivity of the assay for the eight toxins was lower than the oral LD50 which would likely be used in a criminal contamination of food supply. This assay should be useful in monitoring biological threats. In the public‐health domain, it opens the way for multiplex investigation of food‐borne toxins using targeted LC‐MS/MS.     

Application of quantitative proteomics to the integrated analysis of the ubiquitylated and global proteomes of xenograft tumor tissues

Background

Post-translational modification by ubiquitin is a fundamental regulatory mechanism that is implicated in many cellular processes including the cell cycle, apoptosis, cell adhesion, angiogenesis, and tumor growth. The low stoichiometry of ubiquitylation presents an analytical challenge for the detection of endogenously modified proteins in the absence of enrichment strategies. The recent availability of antibodies recognizing peptides with Lys residues containing a di-Gly ubiquitin remnant (K-ε-GG) has greatly improved the ability to enrich and identify ubiquitylation sites from complex protein lysates via mass spectrometry. To date, there have not been any published studies that quantitatively assess the changes in endogenous ubiquitin-modification protein stoichiometry status at the proteome level from different tissues.

Results

In this study, we applied an integrated quantitative mass spectrometry based approach using isobaric tags for relative and absolute quantitation (iTRAQ) to interrogate the ubiquitin-modified proteome and the cognate global proteome levels from luminal and basal breast cancer patient-derived xenograft tissues. Among the proteins with quantitative global and ubiquitylation data, 91 % had unchanged levels of total protein relative abundance, and less than 5 % of these proteins had up- or down-regulated ubiquitylation levels. Of particular note, greater than half of the proteins with observed changes in their total protein level also had up- or down-regulated changes in their ubiquitylation level.

Conclusions

This is the first report of the application of iTRAQ-based quantification to the integrated analysis of the ubiquitylated and global proteomes at the tissue level. Our results underscore the importance of conducting integrated analyses of the global and ubiquitylated proteomes toward elucidating the specific functional significance of ubiquitylation.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9086-5) contains supplementary material, which is available to authorized users.     

Accurate mass comparison coupled with two endopeptidases enables identification of protein termini

Protein termini play important roles in biological processes, but there have been few methods for comprehensive terminal proteomics. We have developed a new method that can identify both the amino and the carboxyl termini of proteins. The method independently uses two proteases, (lysyl endopeptidase) Lys-C and peptidyl-Lys metalloendopeptidase (Lys-N), to digest proteins, followed by LC-MS/MS analysis of the two digests. Terminal peptides can be identified by comparing the peptide masses in the two digests as follows: (i) the amino terminal peptide of a protein in Lys-C digest is one lysine residue mass heavier than that in Lys-N digest; (ii) the carboxyl terminal peptide in Lys-N digest is one lysine residue mass heavier than that in Lys-C digest; and (iii) all internal peptides give exactly the same molecular masses in both the Lys-C and the Lys-N digest, although amino acid sequences of Lys-C and Lys-N peptides are different (Lys-C peptides end with lysine, whereas Lys-N peptides begin with lysine). The identification of terminal peptides was further verified by examining their MS/MS spectra to avoid misidentifying pairs as termini. In this study, we investigated the usefulness of this method using several protein and peptide mixtures. Known protein termini were successfully identified. Acetylation on N-terminus and protein isoforms, which have different termini, was also determined. These results demonstrate that our new method can confidently identify terminal peptides in protein mixtures.     

The role of statistical power analysis in quantitative proteomics

Designing an experiment for quantitative proteomic analysis is not a trivial task. One of the key factors influencing the success of such studies is the number of biological replicates included in the analysis. This, along with the measured variation will determine the statistical power of the analysis. Presented is a simple yet powerful analysis to determine the appropriate sample size required for reliable and reproducible results, based on the total variation (technical and biological). This approach can also be applied retrospectively for the interpretation of results as it takes into account both significance (p value) and quantitative difference (fold change) of the results.     

Building and searching tandem mass (MS/MS) spectral libraries for peptide identification in proteomics

Spectral library searching is an emerging approach in peptide identifications from tandem mass spectra, a critical step in proteomic data analysis. In spectral library searching, a spectral library is first meticulously compiled from a large collection of previously observed peptide MS/MS spectra that are conclusively assigned to their corresponding amino acid sequence. An unknown spectrum is then identified by comparing it to all the candidates in the spectral library for the most similar match. This review discusses the basic principles of spectral library building and searching, describes its advantages and limitations, and provides a primer for researchers interested in adopting this new approach in their data analysis. It will also discuss the future outlook on the evolution and utility of spectral libraries in the field of proteomics.     

A paired ions scoring algorithm based on Morpheus for simultaneous identification and quantification of proteome samples prepared by isobaric peptide termini labeling strategies

The isobaric peptide termini labeling (IPTL) method is a promising strategy in quantitative proteomics for its high accuracy, while the increased complexity of MS2 spectra originated from the paired b, y ions has adverse effect on the identification and the coverage of quantification. Here, a paired ions scoring algorithm (PISA) based on Morpheus, a database searching algorithm specifically designed for high‐resolution MS2 spectra, was proposed to address this issue. PISA was first tested on two 1:1 mixed IPTL datasets, and increases in peptide to spectrum matchings, distinct peptides and protein groups compared to Morpheus itself and MASCOT were shown. Furthermore, the quantification is simultaneously performed and 100% quantification coverage is achieved by PISA since each of the identified peptide to spectrum matchings has several pairs of fragment ions which could be used for quantification. Then the PISA was applied to the relative quantification of human hepatocellular carcinoma cell lines with high and low metastatic potentials prepared by an IPTL strategy.     

Application of quantitative proteomic analysis using tandem mass tags for discovery and identification of novel biomarkers in periodontal disease

Periodontal disease is a bacterial infection that destroys the gingiva and surrounding tissues of the oral cavity. Gingival crevicular fluid (GCF) is extracted from the gingival sulcus and pocket. Analysis of biochemical markers in GCF, which predict the progression of periodontal disease, may facilitate disease diagnosis. However, no useful GCF biochemical markers with high sensitivity for detecting periodontal disease have been identified. Thus, the search for biochemical markers of periodontal disease is of continued interest in experimental and clinical periodontal disease research. Using tandem mass tag (TMT) labeling, we analyzed GCF samples from healthy subjects and patients with periodontal disease, and identified a total of 619 GCF proteins based on proteomic analysis. Of these, we focused on two proteins, matrix metalloproteinase (MMP)‐9 and neutrophil gelatinase‐associated lipocalin (LCN2), which are involved in the progression of periodontal disease. Western blot analysis revealed that the levels of MMP‐9 and LCN2 were significantly higher in patients with periodontal disease than in healthy subjects. In addition, ELISA also detected significantly higher levels of LCN2 in patients with periodontal disease than in healthy subjects. Thus, LC‐MS/MS analyses of GCF using TMT labeling led to the identification of LCN2, which may be a promising GCF biomarker for the detection of periodontal disease.     

Expanding the organismal scope of proteomics: cross-species protein identification by mass spectrometry and its implications

Due to the limited applicability of conventional protein identification methods to the proteomes of organisms with unsequenced genomes, researchers have developed approaches to identify proteins using mass spectrometry and sequence similarity database searches. Both the integration of mass spectrometry with bioinformatics and genomic sequencing drive the expanding organismal scope of proteomics.     

Labeling elastase digests with TMT: informational gain by identification of poorly detectable peptides with MALDI-TOF/TOF mass spectrometry

The applicability of the less specific protease elastase for the identification of membrane and cytosolic proteins has already been demonstrated. MALDI as ionization technique particularly favors the detection of basic and to a lesser extent of weakly acidic peptides, whereas neutral peptides often remain undetected. Moreover, peptides below 700 Da are routinely excluded. In the following study, the advantage of additional information gained from tandem mass tag zero labeled peptides and the resultant increase in sequence coverage was evaluated. Through derivatization with tandem mass tag reagents, peptide measurement within the standard mass range of the MALDI reflector mode is achievable due to the mass increase. Compared to the unlabeled sample, peptides exhibiting relatively low molecular masses, pI values or higher hydrophobicity could be identified.     

Sortase A mediated site‐specific immobilization for identification of protein interactions in affinity purification–mass spectrometry experiments

Proteomics approaches using MS in combination with affinity purification have emerged as powerful tools to study protein‐protein interactions. Here we make use of the specificity of sortase A transpeptidation reaction to prepare affinity matrices in which a protein bait is covalently linked to the matrix via a short C‐terminal linker region. As a result of this site‐directed immobilization, the bait remains functionally accessible to protein interactions. To apply this approach, we performed SILAC‐based pull‐down experiments and demonstrate the suitability of the approach.     

Proteome-wide Analysis of Protein Thermal Stability in the Model Higher Plant Arabidopsis thaliana

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Highlights

• •Over 1700 Arabidopsis proteins with thermal models in multiple replicates.
• •Melting temperature correlates with 1°, 2°, and 3° protein characteristics.
• •Ligand-induced thermal shifts are evident in complex protein extracts.

     

Increasing the mass accuracy of high-resolution LC-MS data using background ions: a case study on the LTQ-Orbitrap

With the advent of a new generation of high-resolution mass spectrometers, the fields of proteomics and metabolomics have gained powerful new tools. In this paper, we demonstrate a novel computational method that improves the mass accuracy of the LTQ-Orbitrap mass spectrometer from an initial +/- 1-2 ppm, obtained by the standard software, to an absolute median of 0.21 ppm (SD 0.21 ppm). With the increased mass accuracy it becomes much easier to match mass chromatograms in replicates and different sample types, even if compounds are detected at very low intensities. The proposed method exploits the ubiquitous presence of background ions in LC-MS profiles for accurate alignment and internal mass calibration, making it applicable for all types of MS equipment. The accuracy of this approach will facilitate many downstream systems biology applications, including mass-based molecule identification, ab initio metabolic network reconstruction, and untargeted metabolomics in general.     

Analysis of thyroglobulin iodination by tandem mass spectrometry using immonium ions of monoiodo- and diiodo-tyrosine

Peptides containing a monoiodo- or diiodo-tyrosine residue (monoiodo-Y, diiodo-Y) were found to generate abundant immonium ions following collision-induced dissociation at m/z 261.97 and 387.87 Da, respectively. These residue-specific marker ions are between about 140 mDa (monoiodo-Y) and 300 mDa (diiodo-Y) mass deficient relative to any other peptide fragment ions of unmodified peptides, qualifying them as highly specific marker ions for tyrosine iodination when analyzed by high resolution tandem mass spectrometry (MS/MS). Two new iodination sites (Y-364 and Y-2165) were pinpointed in bovine thyroglobulin by MS/MS using these iodotyrosine-specific marker ions and combined tryptic/chymotryptic digestion.     

Prospective applications of ultrahigh resolution proteomics in clinical mass spectrometry

Introduction: Advances in mass spectrometry (MS)-based proteomic strategies have resulted in robust protein biomarker discovery studies often performed on high resolution accurate mass (HRAM) platforms. For successful translation of promising protein biomarkers into useful clinical tests, trans-sector networks and collaboration among stakeholders involved in the biomarker pipeline are urgently needed.

Areas covered: In this perspective, literature- and empirical evidence is combined with author’s opinions to discuss the progress of ultrahigh resolution MS and provide insight in its potential for validation and development of clinical tests.

Expert commentary: Thus far two ‘low resolution’ MS strategies have been implemented in the clinic: quantification of proteins using triple quadrupole instruments and identification of unknown microorganisms using comparative analysis with spectral libraries on MALDI-TOF instruments. The rise of HRAM technology further boosts the potential of MS-based tests for detection and quantitation of disease-specific biomarkers which meet the analytical performance specifications needed for clinical assays.     

Mass spectrometric identification of novel posttranslational modification sites in Huntingtin

Huntington's disease (HD) is caused by a CAG triplet repeat expansion in exon 1 of the Huntingtin (Htt) gene, encoding an abnormal expanded polyglutamine (polyQ) tract that confers toxicity to the mutant Htt (mHtt) protein. Recent data suggest that posttranslational modifications of mHtt modulate its cytotoxicity. To further understand the cytotoxic mechanisms of mHtt, we have generated HEK293 cell models stably expressing Strep- and FLAG-tagged Htt containing either 19Q (wild-type Htt), 55Q (mHtt), or 94Q (mHtt) repeats. Following tandem affinity purification, the tagged Htt and associated proteins were subjected to tandem mass spectrometry or 2D nano-LC tandem mass spectrometry and several novel modification sites of mHtt containing 55Q or 94Q were identified. These were phosphorylation sites located at Ser431 and Ser432, and ubiquitination site located at Lys444. The two phosphorylation sites were confirmed by Western blot analysis using phosphorylation site-specific antibodies. In addition, prevention of phosphorylation at the two serine sites altered mHtt toxicity and accumulation. These modifications of mHtt may provide novel therapeutic targets for effective treatment of the disorder.     

Determination of carbon labeling distribution of intracellular metabolites from single fragment ions by ion chromatography tandem mass spectrometry

Liquid chromatography tandem mass spectrometry coupling is a highly sensitive and specific technique allowing molecule detection in the femtomolar range. This article introduces a straightforward approach to apply this technique in 13C metabolic flux analysis. Based on a theoretical analysis of the correlation between molecule ions and corresponding fragments, a method was developed to determine the carbon labeling of intracellular metabolites without increasing the number of measurements per metabolite compared with direct molecule ion analysis. The method was applied to phosphorylated metabolites because their fragmentation results in high yields of [PO3]- and/or [H2PO4]- ions. Comparing the accuracy of the carbon labeling determination of phosphorylated metabolites between direct analysis of the molecule ions with that of corresponding phosphate fragment ions, it could be demonstrated that the introduced approach resulted in significantly higher accuracy and sensitivity for all tested metabolites. When applying the techniques to Escherichia coli cell extracts, 2 microg cell dry weight per injection was sufficient to determine the natural abundances of the carbon fractions m and m+1 from six phosphorylated metabolites with high accuracy, predestining the approach for very small cultivation volumes in the microliter range.     

Isotopologue distributions of peptide product ions by tandem mass spectrometry: quantitation of low levels of deuterium incorporation

Protonated molecular peptide ions and their product ions generated by tandem mass spectrometry appear as isotopologue clusters due to the natural isotopic variations of carbon, hydrogen, nitrogen, oxygen, and sulfur. Quantitation of the isotopic composition of peptides can be employed in experiments involving isotope effects, isotope exchange, and isotopic labeling by chemical reactions and in studies of metabolism by stable isotope incorporation. Both ion trap and quadrupole-time of flight mass spectrometry are shown to be capable of determining the isotopic composition of peptide product ions obtained by tandem mass spectrometry with both precision and accuracy. Tandem mass spectra of clusters of isotopologue ions obtained in profile mode are fit by nonlinear least squares to a series of Gaussian peaks which quantify the Mn/M0 values which define the isotopologue distribution (ID). To determine the isotopic composition of product ions from their ID, a new algorithm that predicts the Mn/M0 ratios and obviates the need to determine the intensity of all of the ions of an ID is developed. Consequently a precise and accurate determination of the isotopic composition of a product ion may be obtained from only the initial values of the ID, however, the entire isotopologue cluster must be isolated prior to fragmentation. Following optimization of the molecular ion isolation width, fragmentation energy, and detector sensitivity, the presence of isotopic excess (2H, 13C, 15N, 18O) is readily determined within 1%. The ability to determine the isotopic composition of sequential product ions permits the isotopic composition of individual amino acid residues in the precursor ion to be determined.