Quantitative LC-MS/MS assays were designed for tryptic peptides representing 53 high and medium abundance proteins in human plasma using a multiplexed multiple reaction monitoring (MRM) approach. Of these, 47 produced acceptable quantitative data, demonstrating within-run coefficients of variation (CVs) (n ؍ 10) of 2-22% (78% of assays had CV <10%). A number of peptides gave CVs in the range 2-7% in five experiments (10 replicate runs each) continuously measuring 137 MRMs, demonstrating the precision achievable in complex digests. Depletion of six high abundance proteins by immunosubtraction significantly improved CVs compared with whole plasma, but analytes could be detected in both sample types. Replicate digest and depletion/digest runs yielded correlation coefficients (R Accurate quantitation of proteins and peptides in plasma and serum is a challenging problem because of the complexity and extreme dynamic range that characterize these samples (1). The widely adopted separative (survey) approach to proteomics, in which an attempt is made to detect all components, has proven to be limited in sensitivity toward low abundance proteins (2) and typically provides limited quantitative precision. The alternative candidate-based approach, which relies on specific assays optimized for quantitative detection of selected proteins, can provide significantly increased sensitivity (into the pg/ml range) and precision (CVs 1 Ͻ 5-10%) at the cost of restricting discovery potential toward novel proteins (3). In practice, a combination of these approaches (one or more survey approaches for de novo biomarker discovery coupled with a candidate-based approach to biomarker validation in large sample sets) appears to provide an effective staged pipeline (4) for generation of valid plasma biomarkers of disease, risk, and therapeutic response.Candidate-based specific assays rely on the specificity of capture or detection methods to select a specific molecule as analyte. Capture reagents such as antibodies can provide extreme specificity (particularly when two different antibodies are used as in a sandwich immunoassay) and form the basis of most existing clinical protein assays. There is intense interest in miniaturizing sets of such assays (5, 6) in array formats (on planar substrates, beads, etc.), although significant problems remain in the production of suitable antibodies and in the simultaneous optimization of multiple assays in one fluid volume.Mass spectrometry provides an alternative assay approach, relying on the discriminating power of mass analyzers to select a specific analyte and on ion current measurements for quantitation. In the field of analytical chemistry, many small molecule analytes (e.g. drug metabolites (7), hormones (8), protein degradation products (9), and pesticides (10)) are routinely measured using this approach at high throughput with great precision (CV Ͻ 5%). Most such assays use electrospray ionization followed by two stages of mass selection: a first stage (MS1) selecting the mass of the intact an...
The Paragon TM Algorithm, a novel database search engine for the identification of peptides from tandem mass spectrometry data, is presented. Sequence Temperature Values are computed using a sequence tag algorithm, allowing the degree of implication by an MS/MS spectrum of each region of a database to be determined on a continuum. Counter to conventional approaches, features such as modifications, substitutions, and cleavage events are modeled with probabilities rather than by discrete user-controlled settings to consider or not consider a feature. The use of feature probabilities in conjunction with Sequence Temperature Values allows for a very large increase in the effective search space with only a very small increase in the actual number of hypotheses that must be scored. The algorithm has a new kind of user interface that removes the user expertise requirement, presenting control settings in the language of the laboratory that are translated to optimal algorithmic settings. To validate this new algorithm, a comparison with Mascot is presented for a series of analogous searches to explore the relative impact of increasing search space probed with Mascot by relaxing the tryptic digestion conformance requirements from trypsin to semitrypsin to no enzyme and with the Paragon Algorithm using its Rapid mode and Thorough mode with and without tryptic specificity. Although they performed similarly for small search space, dramatic differences were observed in large search space. With the Paragon Algorithm, hundreds of biological and artifact modifications, all possible substitutions, and all levels of conformance to the expected digestion pattern can be searched in a single search step, yet the typical cost in search time is only 2-5 times that of conventional small search space. Despite this large increase in effective search space, there is no drastic loss of discrimination that typically accompanies the exploration of large search space.
Quantitative proteomics employing mass spectrometry is an indispensable tool in life science research. Targeted proteomics has emerged as a powerful approach for reproducible quantification but is limited in the number of proteins quantified. SWATH-mass spectrometry consists of data-independent acquisition and a targeted data analysis strategy that aims to maintain the favorable quantitative characteristics (accuracy, sensitivity, and selectivity) of targeted proteomics at large scale. While previous SWATH-mass spectrometry studies have shown high intra-lab reproducibility, this has not been evaluated between labs. In this multi-laboratory evaluation study including 11 sites worldwide, we demonstrate that using SWATH-mass spectrometry data acquisition we can consistently detect and reproducibly quantify >4000 proteins from HEK293 cells. Using synthetic peptide dilution series, we show that the sensitivity, dynamic range and reproducibility established with SWATH-mass spectrometry are uniformly achieved. This study demonstrates that the acquisition of reproducible quantitative proteomics data by multiple labs is achievable, and broadly serves to increase confidence in SWATH-mass spectrometry data acquisition as a reproducible method for large-scale protein quantification.
Liquid chromatography coupled to tandem mass spectrometry is the main method for high-throughput identification and quantification of peptides and inferred proteins. Within this field, data-independent acquisition (DIA) combined with peptide-centric scoring, exemplified by SWATH-MS, emerged as a scalable method to achieve deep and consistent proteome coverage across large-scale datasets. Here we discuss the adaptation of statistical concepts developed for discovery proteomics based on spectrum-centric scoring to large-scale DIA experiments analyzed with peptide-centric scoring strategies and provide guidance on their application. We show that optimal tradeoffs between sensitivity and specificity require careful considerations of the relationship between proteins in the samples and proteins represented in the spectral library. We propose the application of a global analyte constraint to prevent accumulation of false positives across large-scale datasets. Furthermore, to increase the quality and reproducibility of published proteomic results, well-established confidence criteria should be reported for detected peptide queries, peptides and inferred proteins.
We report the design and total chemical synthesis of "synthetic erythropoiesis protein" (SEP), a 51-kilodalton protein-polymer construct consisting of a 166-amino-acid polypeptide chain and two covalently attached, branched, and monodisperse polymer moieties that are negatively charged. The ability to control the chemistry allowed us to synthesize a macromolecule of precisely defined covalent structure. SEP was homogeneous as shown by high-resolution analytical techniques, with a mass of 50,825 +/-10 daltons by electrospray mass spectrometry, and with a pI of 5.0. In cell and animal assays for erythropoiesis, SEP displayed potent biological activity and had significantly prolonged duration of action in vivo. These chemical methods are a powerful tool in the rational design of protein constructs with potential therapeutic applications.
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