Multiplexed absolute quantification for proteomics using concatenated signature peptides encoded by QconCAT genes

An important area of proteomics involves the need for quantification, whether relative or absolute. Many methods now exist for relative quantification, but to support biomarker proteomics and systems biology, absolute quantification rather than relative quantification is required. Absolute quantification usually involves the concomitant mass spectrometric determination of signature proteotypic peptides and stable isotope-labeled analogs. However, the availability of standard labeled signature peptides in accurately known amounts is a limitation to the widespread adoption of this approach. We describe the design and synthesis of artificial QconCAT proteins that are concatamers of tryptic peptides for several proteins. This protocol details the methods for the design, expression, labeling, purification, characterization and use of the QconCATs in the absolute quantification of complex protein mixtures. The total time required to complete this protocol (from the receipt of the QconCAT expression plasmid to the absolute quantification of the set of proteins encoded by the QconCAT protein in an analyte sample) is approximately 29 d.     

IQcat: multiplexed protein quantification by isoelectric QconCAT

Expression of isotopically labeled peptide standards as artificial concatamers (QconCATs) allows for the multiplex quantification of proteins in unlabeled samples by mass spectrometry. We have developed a generalizable QconCAT design strategy, which we term IQcat, wherein concatenated peptides are binned by pI to facilitate MS-sample enrichment by isoelectric focusing. Our method utilizes a rapid (～2 weeks), inexpensive and scalable purification of arg/lys labeled IQcat standards in the Escherichia coli auxotroph AT713. With this pipeline, we assess the fidelity of IQcat-based absolute quantification for ten yeast proteins over a broad concentration range in a single information-rich isoelectric fraction. The technique is further employed for a quantitative study of androgen-dependent protein expression in cultured prostate cancer cells.     

The absolute quantification strategy: a general procedure for the quantification of proteins and post-translational modifications

Advances in biological mass spectrometry have resulted in the development of numerous strategies for the large-scale quantification of protein expression levels within cells. These measurements of protein expression are most commonly accomplished through differential incorporation of stable isotopes into cellular proteins. Several variations of the stable isotope quantification method have been demonstrated, differing in isotope composition and incorporation strategy. In general, the majority of these methods establish only relative quantification of expressed proteins. To address this, the absolute quantification (AQUA) strategy was developed for the precise determination of protein expression and post-translational modification levels. The AQUA method relies on the use of a synthetic internal standard peptide that is introduced at a known concentration to cell lysates during digestion. This AQUA peptide precisely mimics a peptide produced during proteolysis of the target protein, except that it is enriched in certain stable isotopes. Analysis of the proteolyzed sample by a selected reaction monitoring (SRM) experiment in a tandem mass spectrometer results in the direct detection and quantification of both the native peptide and isotope labeled AQUA internal standard peptide. As an example, the development and application of a method to measure a tryptic peptide representing the amount of polyubiquitin chain formation through lysine 48 (K48) is presented. The simplicity and sensitivity of the method, coupled with the widespread availability of tandem mass spectrometers, make the AQUA strategy a highly useful procedure for measuring the levels of proteins and post-translational modifications directly from cell lysates.     

Relative and absolute quantification of postsynaptic density proteome isolated from rat forebrain and cerebellum

The postsynaptic density (PSD) of central excitatory synapses is essential for postsynaptic signaling, and its components are heterogeneous among different neuronal subtypes and brain structures. Here we report large scale relative and absolute quantification of proteins in PSDs purified from adult rat forebrain and cerebellum. PSD protein profiles were determined using the cleavable ICAT strategy and LC-MS/MS. A total of 296 proteins were identified and quantified with 43 proteins exhibiting statistically significant abundance change between forebrain and cerebellum, indicating marked molecular heterogeneity of PSDs between different brain regions. Moreover we utilized absolute quantification strategy, in which synthetic isotope-labeled peptides were used as internal standards, to measure the molar abundance of 32 key PSD proteins in forebrain and cerebellum. These data confirm the abundance of calcium/calmodulin-dependent protein kinase II and PSD-95 and reveal unexpected stoichiometric ratios between glutamate receptors, scaffold proteins, and signaling molecules in the PSD. Our data also demonstrate that the absolute quantification method is well suited for targeted quantitative proteomic analysis. Overall this study delineates a crucial molecular difference between forebrain and cerebellar PSDs and provides a quantitative framework for measuring the molecular stoichiometry of the PSD.     

Global analysis of a "simple" proteome: Methanococcus jannaschii

The completed genome of Methanococcus jannaschii, including the main chromosome and two extra-chromosomal elements, predicts a proteome comprised of 1783 proteins. How many of those proteins are expressed at any given time and the relative abundance of the expressed proteins, however, cannot be predicted solely from the genome sequence. Two-dimensional gel electrophoresis coupled with peptide mass spectrometry is being used to identify the proteins expressed by M. jannaschii cells grown under different conditions as part of an effort to correlate protein expression with regulatory mechanisms. Here we describe the identification of 170 of the most abundant proteins found in total lysates of M. jannaschii grown under optimal fermentation conditions. To optimize the number of proteins detected, two different protein specific stains (Coomassie Blue R250 or silver nitrate) and two different first dimension separation methods (isoelectric focusing or nonequilibrium pH gradient electrophoresis) were used. Thirty-two percent of the proteins identified are annotated as hypothetical (21% conserved hypothetical and 11% hypothetical), 21% are enzymes involved in energy metabolism, 12% are proteins required for protein synthesis, and the remainder include proteins necessary for intermediary metabolism, cell division, and cell structure. Evidence of post-translational modification of numerous M. jannaschii proteins has been found, as well as indications of incomplete dissociation of protein-protein complexes. These results demonstrate the complexity of proteome analysis even when dealing with a relatively simple genome.     

The importance of the digest: proteolysis and absolute quantification in proteomics

Virtually all mass spectrometric-based methods for quantitative proteomics are at the peptide level, whether label-mediated or label-free. Absolute quantification in particular is based on the measurement of limit peptides, defined as those peptides that cannot be further fragmented by the protease in use. Complete release of analyte and (stable isotope labelled) standard ensures that the most reliable quantification data are recovered, especially when the standard peptides are in a different primary sequence context, such as sometimes occurs in the QconCAT methodology. Moreover, in label-free methods, incomplete digestion would diminish the ion current attributable to limit peptides and lead to artifactually low quantification data. It follows that an essential requirement for peptide-based absolute quantification in proteomics is complete and consistent proteolysis to limit peptides. In this paper we describe strategies to assess completeness of proteolysis and discuss the potential for variance in digestion efficiency to compromise the ensuing quantification data. We examine the potential for kinetically favoured routes of proteolysis, particularly at the last stages of the digestion, to direct products into ‘dead-end’ mis-cleaved products.     

Methods for the absolute quantification of N-glycan biomarkers

BackgroundMany treatment options especially for cancer show a low efficacy for the majority of patients demanding improved biomarker panels for patient stratification. Changes in glycosylation are a hallmark of many cancers and inflammatory diseases and show great potential as clinical disease markers. The large inter-subject variability in glycosylation due to hereditary and environmental factors can complicate rapid transfer of glycan markers into the clinical practice but also presents an opportunity for personalized medicine.Scope of reviewThis review discusses opportunities of glycan biomarkers in personalized medicine and reviews the methodology for N-glycan analysis with a specific focus on methods for absolute quantification.Major conclusionsThe entry into the clinical practice of glycan markers is delayed in large part due to a lack of adequate methodology for the precise and robust quantification of protein glycosylation. Only absolute glycan quantification can provide a complete picture of the disease related changes and will provide the method robustness required by clinical applications.General significanceGlycan biomarkers have a huge potential as disease markers for personalized medicine. The use of stable isotope labeled glycans as internal standards and heavy-isotope labeling methods will provide the necessary method precision and robustness acceptable for clinical use. This article is part of a Special Issue entitled “Glycans in personalized medicine” Guest Editor: Professor Gordan Lauc.     

Global impact of oncogenic Src on a phosphotyrosine proteome

Elevated activity of Src, the first characterized protein-tyrosine kinase, is associated with progression of many human cancers, and Src has attracted interest as a therapeutic target. Src is known to act in various receptor signaling systems to impact cell behavior, yet it remains likely that the spectrum of Src protein substrates relevant to cancer is incompletely understood. To better understand the cellular impact of deregulated Src kinase activity, we extensively applied a mass spectrometry shotgun phosphotyrosine (pTyr) proteomics strategy to obtain global pTyr profiles of Src-transformed mouse fibroblasts as well as their nontransformed counterparts. A total of 867 peptides representing 563 distinct pTyr sites on 374 different proteins were identified from the Src-transformed cells, while 514 peptides representing 275 pTyr sites on 167 proteins were identified from nontransformed cells. Distinct characteristics of the two profiles were revealed by spectral counting, indicative of pTyr site relative abundance, and by complementary quantitative analysis using stable isotope labeling with amino acids in cell culture (SILAC). While both pTyr profiles are replete with sites on signaling and adhesion/cytoskeletal regulatory proteins, the Src-transformed profile is more diverse with enrichment in sites on metabolic enzymes and RNA and protein synthesis and processing machinery. Forty-three pTyr sites (32 proteins) are predicted as major biologically relevant Src targets on the basis of frequent identification in both cell populations. This select group, of particular interest as diagnostic biomarkers, includes well-established Src sites on signaling/adhesion/cytoskeletal proteins, but also uncharacterized sites of potential relevance to the transformed cell phenotype.     

Towards proteome standards: The use of absolute quantitation in high-throughput biomarker discovery

The use of proteomics to profile biological fluids and identify therein biomarkers for cancer and other diseases was initially received with considerable excitement. However, results have fallen short of the expectations. Traditionally, protein biomarkers have been identified by measurement of relative expression changes between case and control samples from which differentially expressed proteins are then considered to represent biomarker candidates.We argue that current individual proteomics-based biomarker discovery studies lack the statistical strength for the identification of high-confidence biomarkers. Instead, multi-group efforts are necessary to facilitate the generation of sufficient sample sizes. This is contingent on the ability to collate and cross-compare data from different studies, which will require the use of a common metric or standards.Though profound, the technical challenges for absolute protein quantification can be overcome. The use of matrix specific, shared standards for absolute quantitation presents an opportunity to facilitate the much needed, but currently impossible, comparisons of different studies. In addition to community-wide approaches to standardize pre-analytical biomarker research studies, it is also important to establish means to integrate experimental data from different studies in order to assess the usefulness of proposed biomarkers with sufficient statistical certainty.     

Global proteome quantification for discovering imatinib-induced perturbation of multiple biological pathways in K562 human chronic myeloid leukemia cells

Imatinib mesylate, currently marketed by Novartis as Gleevec in the U.S., has emerged as the leading compound to treat the chronic phase of chronic myeloid leukemia (CML), through its inhibition of Bcr-Abl tyrosine kinases, and other cancers. However, resistance to imatinib develops frequently, particularly in late-stage disease. To identify new cellular pathways affected by imatinib treatment, we applied mass spectrometry together with stable isotope labeling by amino acids in cell culture (SILAC) for the comparative study of protein expression in K562 cells that were untreated or treated with a clinically relevant concentration of imatinib. Our results revealed that, among the 1344 quantified proteins, 73 had significantly altered levels of expression induced by imatinib and could be quantified in both forward and reverse SILAC labeling experiments. These included the down-regulation of thymidylate synthase, S-adenosylmethionine synthetase, and glycerol-3-phosphate dehydrogenase as well as the up-regulation of poly(ADP-ribose) polymerase 1, hemoglobins, and enzymes involved in heme biosynthesis. We also found, by assessing alteration in the acetylation level in histone H4 upon imatinib treatment, that the imatinib-induced hemoglobinization and erythroid differentiation in K562 cells are associated with global histone H4 hyperacetylation. Overall, these results provided potential biomarkers for monitoring the therapeutic intervention of CML using imatinib and offered important new knowledge for gaining insight into the molecular mechanisms of action of imatinib.     

Global Analysis of the Hortaea werneckii proteome: studying steroid response in yeast

The response of the halophilic black yeast Hortaea werneckii to the steroid hormone progesterone has been studied at the protein level using fluorescent two-dimensional differential gel electrophoresis (2D-DIGE) technology in combination with mass spectrometry. Data on protein identification from this study reveal molecular mechanisms of the response to progesterone. In particular, the overexpression of Pck2 and Pac2 in the stimulated cells indicates the interactions of progesterone with the cell growth and reproduction signaling pathways.     

A strategy to investigate the plant meiotic proteome

The analysis of meiosis in higher plants has benefited considerably in recent years from the completion of the genome sequence of the model plant Arabidopsis thaliana and the development of cytological techniques for this species. A combination of forward and reverse genetics has provided important routes toward the identification of meiotic genes in Arabidopsis. Nevertheless identification of certain meiotic genes remains a challenge due to problems such as limited sequence conservation between species, existence of closely related gene families and in some cases functional redundancy between gene family members. Hence there is a requirement to develop new experimental approaches that can be used in conjunction with existing methods to enable a greater range of plant meiotic genes to be identified. As one potential route towards this goal we have initiated a proteomics-based approach. Unfortunately, the small size of Arabidopsis anthers makes an analysis in this species technically very difficult. Therefore we have initially focussed on Brassica oleracea which is closely related to Arabidopsis, but has the advantage of possessing significantly larger anthers. The basic strategy has been to use peptide mass-finger printing and matrix-assisted laser desorption ionization time of flight mass spectrometry to analyse proteins expressed in meiocytes during prophase I of meiosis. Initial experiments based on the analysis of proteins from staged anther tissue proved disappointing due to the low level of detection of proteins associated with meiosis. However, by extruding meiocytes in early prophase I from individual anthers prior to analysis a significant enrichment of meiotic proteins has been achieved. Analysis suggests that at least 18% of the proteins identified by this route have a putative meiotic function and that this figure could be as high as one-third of the total. Approaches to increase the enrichment of proteins involved in meiotic recombination and chromosome synapsis are also described.     

Use of a proteome strategy for tagging proteins present at the plasma membrane

A plasma membrane (PM) fraction was purified from Arabidopsis thaliana using a standard procedure and analyzed by two-dimensional (2D) gel electrophoresis. The proteins were classified according to their relative abundance in PM or cell membrane supernatant fractions. Eighty-two of the 700 spots detected on the PM 2D gels were microsequenced. More than half showed sequence similarity to proteins of known function. Of these, all the spots in the PM-specific and PM-enriched fractions, together with half of the spots with similar abundance in PM fraction and supernatant, have previously been found at the PM, supporting the validity of this approach. Extrapolation from this analysis indicates that (i) approximately 550 polypeptides found at the PM could be resolved on 2D gels; (ii) that numerous proteins with multiple locations are found at the PM; and (iii) that approximately 80% of PM-specific spots correspond to proteins with unknown function. Among the later, half are represented by ESTs or cDNAs in databases. In this way, several unknown gene products were potentially localized to the PM. These data are discussed with respect to the efficiency of organelle proteome approaches to link systematically genomic data to genome expression. It is concluded that generalized proteomes can constitute a powerful resource, with future completion of Arabidopsis genome sequencing, for genome-wide exploration of plant function.     

Global relative and absolute quantitation in microbial proteomics

Proteomic studies are designed to yield either qualitative information on proteins (identification, distribution, posttranslational modifications, interactions, structure and function) or quantitative information (abundance, distribution within different localizations, temporal changes in abundance due to synthesis and degradation or both). To this end these studies can draw upon a wide range of qualitative and quantitative gel-based and gel-free techniques. This review summarizes current proteomic workflows for global relative or absolute protein quantitation and their application in microbial physiology.     

Positional proteomics: preparation of amino-terminal peptides as a strategy for proteome simplification and characterization

We describe a protocol for selective extraction of the amino (N)-terminal-most peptide of a protein or a mixture of proteins after proteolysis. The first stage of the protocol blocks the free amino groups alpha and epsilon (the latter being lysyl residues) on the intact proteins by acetylation. In the second stage, proteolysis of the acetylated proteins yields a mixture of N-terminally acetylated (true N-terminal) and non-acetylated (internal and carboxy-terminal) peptides. Affinity capture of peptides bearing free amino groups using an immobilized amine-reactive reagent removes internal peptides from the mixture. The unbound fraction is highly enriched in N-terminal peptides, which can be analyzed without further treatment. This method is compatible with a range of proteolytic enzymes and fragmentation methods, and should take 2 d to complete. The N-terminal peptides can then be analyzed by mass spectrometry. This low cost, rapid method is readily adopted using off the shelf reagents.