Mass spectrometry-based quantitative proteomics

A major aim of present-day proteomics is to study changes in protein expression levels at a global level, ideally monitoring all proteins present in cells or tissue. Mass spectrometry is a well-respected technology in proteomics that is widely used for the identification of proteins. More recently, methodologies have been introduced showing that mass spectrometry can also be used for protein quantification. This article reviews various mass spectrometry-based technologies in quantitative proteomics, highlighting several interesting applications in areas ranging from cell biology to clinical applications.     

Mass spectrometry-based quantitative proteomics

A major aim of present-day proteomics is to study changes in protein expression levels at a global level, ideally monitoring all proteins present in cells or tissue. Mass spectrometry is a well-respected technology in proteomics that is widely used for the identification of proteins. More recently, methodologies have been introduced showing that mass spectrometry can also be used for protein quantification. This article reviews various mass spectrometry-based technologies in quantitative proteomics, highlighting several interesting applications in areas ranging from cell biology to clinical applications.     

Mass spectrometry-based proteomics for systems biology

Mass spectrometry (MS)-based proteomics has significantly contributed to the development of systems biology, a new paradigm for the life sciences in which biological processes are addressed in terms of dynamic networks of interacting molecules. Because of its advanced analytical capabilities, MS-based proteomics has been used extensively to identify the components of biological systems, and it is the method of choice to consistently quantify the effects of network perturbation in time and space. Herein, we review recent contributions of MS to systems biology and discuss several examples that illustrate the importance of mass spectrometry to elucidate the components and interactions of molecular networks.     

Mass spectrometry-based proteomics turns quantitative

The field of proteomics is built on technologies to analyze large numbers of proteins--ideally the entire proteome--in the same experiment. Mass spectrometry (MS) has been successfully used to characterize proteins in complex mixtures, but results so far have largely been qualitative. Two recently developed methodologies offer the opportunity to obtain quantitative proteomic information. Comparing the signals from the same peptide under different conditions yields a rough estimate of relative protein abundance between two proteomes. Alternatively, and more accurately, peptides are labeled with stable isotopes, introducing a predictable mass difference between peptides from two experimental conditions. Stable isotope labels can be incorporated 'post-harvest', by chemical approaches or in live cells through metabolic incorporation. This isotopic handle facilitates direct quantification from the mass spectra. Using these quantitative approaches, precise functional information as well as temporal changes in the proteome can be captured by MS.     

Mass spectrometry-based immuno-precipitation proteomics - the user's guide

Immuno-precipitation (IP) experiments using MS provide a sensitive and accurate way of characterising protein complexes and their response to regulatory mechanisms. Differences in stoichiometry can be determined as well as the reliable identification of specific binding partners. The quality control of IP and protein interaction studies has its basis in the biology that is being observed. Is that unusual protein identification a genuine novelty, or an experimental irregularity? Antibodies and the solid matrices used in these techniques isolate not only the target protein and its specific interaction partners but also many non-specific 'contaminants' requiring a structured analysis strategy. These methodological developments and the speed and accuracy of MS machines, which has been increasing consistently in the last 5 years, have expanded the number of proteins identified and complexity of analysis. The European Science Foundation's Frontiers in Functional Genomics programme 'Quality Control in Proteomics' Workshop provided a forum for disseminating knowledge and experience on this subject. Our aim in this technical brief is to outline clearly, for the scientists wanting to carry out this kind of experiment, and recommend what, in our experience, are the best potential ways to design an IP experiment, to help identify possible pitfalls, discuss important controls and outline how to manage and analyse the large amount of data generated. Detailed experimental methodologies have been referenced but not described in the form of protocols.     

Mass spectrometry-based proteomics strategies for protease cleavage site identification

Protease-catalyzed hydrolysis of peptide bonds is one of the most pivotal post-translational modifications fulfilling manifold functions in the regulation of cellular processes. Therefore, dysregulation of proteolytic reactions plays a central role in many pathophysiological events. For this reason, understanding the molecular mechanisms in proteolytic reactions, in particular the knowledge of proteases involved in complex processes, expression levels and activity of protease and knowledge of the targeted substrates are an indispensable prerequisite for targeted drug development. The present review focuses on mass spectrometry-based proteomic methods for the analysis of protease cleavage sites, including the identification of the hydrolyzed bonds as well as of the surrounding sequence. Peptide- and protein-centric approaches and bioinformatic tools for experimental data interpretation will be presented and the major advantages and drawbacks of the different approaches will be addressed. The recent applications of these approaches for the analysis of biological function of different protease classes and potential future directions will be discussed.     

Mass spectrometry-based proteomics combined with bioinformatic tools for bacterial classification

Timely classification and identification of bacteria is of vital importance in many areas of public health. We present a mass spectrometry (MS)-based proteomics approach for bacterial classification. In this method, a bacterial proteome database is derived from all potential protein coding open reading frames (ORFs) found in 170 fully sequenced bacterial genomes. Amino acid sequences of tryptic peptides obtained by LC-ESI MS/MS analysis of the digest of bacterial cell extracts are assigned to individual bacterial proteomes in the database. Phylogenetic profiles of these peptides are used to create a matrix of sequence-to-bacterium assignments. These matrixes, viewed as specific assignment bitmaps, are analyzed using statistical tools to reveal the relatedness between a test bacterial sample and the microorganism database. It is shown that, if a sufficient amount of sequence information is obtained from the MS/MS experiments, a bacterial sample can be classified to a strain level by using this proteomics method, leading to its positive identification.     

Mass spectrometry-based proteomics for translational research: a technical overview

Mass spectrometry-based investigation of clinical samples enables the high-throughput identification of protein biomarkers. We provide an overview of mass spectrometry-based proteomic techniques that are applicable to the investigation of clinical samples. We address sample collection, protein extraction and fractionation, mass spectrometry modalities, and quantitative proteomics. Finally, we examine the limitations and further potential of such technologies. Liquid chromatography fractionation coupled with tandem mass spectrometry is well suited to handle mixtures of hundreds or thousands of proteins. Mass spectrometry-based proteome elucidation can reveal potential biomarkers and aid in the development of hypotheses for downstream investigation of the molecular mechanisms of disease.     

Mass spectrometry-based proteomics and peptidomics for systems biology and biomarker discovery

The scientific community has shown great interest in the field of mass spectrometry-based proteomics and peptidomics for its applications in biology.Proteomics technologies have evolved to produce larg...     

Mass spectrometry-based functional proteomics: from molecular machines to protein networks

The study of protein-protein interactions by mass spectrometry is an increasingly important part of post-genomics strategies to understand protein function. A variety of mass spectrometry-based approaches allow characterization of cellular protein assemblies under near-physiological conditions and subsequent assignment of individual proteins to specific molecular machines, pathways and networks, according to an increasing level of organizational complexity. An appropriate analytical strategy can be individually tailored--from an in-depth analysis of single complexes to a large-scale characterization of entire molecular pathways or even an analysis of the molecular organization of entire expressed proteomes. Here we review different options regarding protein-complex purification strategies, mass spectrometry analysis and bioinformatic methods according to the specific question that is being addressed.     

Signal maps for mass spectrometry-based comparative proteomics

Mass spectrometry-based proteomic experiments, in combination with liquid chromatography-based separation, can be used to compare complex biological samples across multiple conditions. These comparisons are usually performed on the level of protein lists generated from individual experiments. Unfortunately given the current technologies, these lists typically cover only a small fraction of the total protein content, making global comparisons extremely limited. Recently approaches have been suggested that are built on the comparison of computationally built feature lists instead of protein identifications. Although these approaches promise to capture a bigger spectrum of the proteins present in a complex mixture, their success is strongly dependent on the correctness of the identified features and the aligned retention times of these features across multiple experiments. In this experimental-computational study, we went one step further and performed the comparisons directly on the signal level. First signal maps were constructed that associate the experimental signals across multiple experiments. Then a feature detection algorithm used this integrated information to identify those features that are discriminating or common across multiple experiments. At the core of our approach is a score function that faithfully recognizes mass spectra from similar peptide mixtures and an algorithm that produces an optimal alignment (time warping) of the liquid chromatography experiments on the basis of raw MS signal, making minimal assumptions on the underlying data. We provide experimental evidence that suggests uniqueness and correctness of the resulting signal maps even on low accuracy mass spectrometers. These maps can be used for a variety of proteomic analyses. Here we illustrate the use of signal maps for the discovery of diagnostic biomarkers. An imple-mentation of our algorithm is available on our Web server.     

Liquid chromatography-mass spectrometry-based quantitative proteomics

LC-MS-based quantitative proteomics has become increasingly applied to a wide range of biological applications due to growing capabilities for broad proteome coverage and good accuracy and precision in quantification. Herein, we review the current LC-MS-based quantification methods with respect to their advantages and limitations and highlight their potential applications.     

Modified cysteine S-phosphopeptide standards for mass spectrometry-based proteomics

The regulatory role of protein cysteine phosphorylation is an under-researched area. The difficulty of accessing reference S-phosphorylated peptides (pCys-peptides) hampers progress in MS-driven cysteine phosphoproteomics, which requires targeted analytical procedures. This work describes an uncomplicated process for the conversion of disulfide-bridged protein into a complex model mixture of combinatorially modified peptides. Hen egg-white lysozyme was reduced with tris(2-carboxyethyl)phosphine (TCEP) followed by alkylation of cysteine with (3-acrylamidopropyl)trimethyl-ammonium chloride (APTA) and subsequent beta-elimination in aqueous Ba(OH)₂ to yield modified polypeptides containing multiple dehydroalanine (Dha) residues. The conjugate addition of thiophosphoric acid to Dha residues followed by trypsinolysis led to numerous D/L phosphocysteine-containing peptides, which were identified by higher-energy collisional-dissociation tandem mass spectrometry (HCD-MS/MS). Our results show that some pCys-peptides produce prominent neutral losses of 80 Da, 98 Da and a weak 116 Da loss. These are similar to the neutral-loss triplets generated by phosphohistidine peptides.