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.

IntroductionThe key event in the pathogenesis of arteriosclerosis is believed to be a dysfunction of the endothelium with disruption of vascular homeostasis, predisposing blood vessels to vasoconstriction, inflammation, leukocyte adhesion, thrombosis, and proliferation of vascular smooth muscle cells. Red blood cells (RBCs) are typically considered as shuttles of respiratory gases and nutrients for tissues, less so compartments important to vascular integrity. Patients with coronary artery disease (CAD) and concomitant anemia have a poorer prognosis after myocardial infarction, percutaneous coronary intervention, and coronary artery bypass grafting, and are more prone to developing heart failure with fatal outcomes. [1][2][3] Surprisingly, erythropoietin treatment fails to improve diagnosis, indicating that a compromised gas exchange/nutrient transport capacity of blood is insufficient to explain this outcome.Nitric oxide (NO) is an essential short-lived signaling/ regulatory product of a healthy endothelium that is critically important for vascular health. Decreased production and/or bioactivity of NO are a hallmark of endothelial dysfunction and have been shown to contribute to accelerated atherogenesis. In the cardiovascular system, NO is continuously produced in endothelial cells (ECs) by the type III isoform of NO synthase (eNOS, NOS3; EC 1.14.13.39). 4 In addition to endothelial cells, some circulating blood cells also contain eNOS.It is an accepted dogma that RBCs take up and inactivate endothelium-derived NO via rapid reaction with oxyhemoglobin to form methemoglobin and nitrate, thereby limiting NO available for vasodilatation. Yet it has also been shown that RBCs not only act as "NO sinks" but synthesize, store, and transport NO metabolic products. Under hypoxic conditions in particular, it has been demonstrated that RBCs induce NO-dependent vasorelaxation. 5,6 Mechanisms of release and potential sources of NO in RBCs are still a matter of debate, but candidates include iron-nitrosylhemoglobin, 7 S-nitrosohemoglobin, [8][9][10] and nitrite. The latter may form NO either via deoxyhemoglobin 5,11 or xanthine oxidoreductase (XOR)-mediated reduction, 6,12 or via spontaneous 12 and carbonic anhydrase-facilitated disproportionation. 13 Most of these processes show a clear oxygen-dependence, and several are favored by low oxygen tensions. The relative contribution of either mechanism to NO formation varies with oxygen partial pressure along the vascular tree. In addition, RBCs release ATP when subjected to hypoxia, providing an alternative vasodilatory pathway. 14 The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. 16,19 and citrulline 15,18 in the supernatant. However, Kang et al failed to measure citrulline production in RBC lysates, 20 maybe because of loss of cellular structures or cofactors important for activity. 21 Another recent study fai...

The use of data-independent acquisition methods such as SWATH for mass spectrometry based proteomics is usually performed with peptide MS/MS assay libraries which enable identification and quantitation of peptide peak areas. Reference assay libraries can be generated locally through information dependent acquisition, or obtained from community data repositories for commonly studied organisms. However, there have been no studies performed to systematically evaluate how locally generated or repository-based assay libraries affect SWATH performance for proteomic studies. To undertake this analysis, we developed a software workflow, SwathXtend, which generates extended peptide assay libraries by integration with a local seed library and delivers statistical analysis of SWATH-quantitative comparisons. We designed test samples using peptides from a yeast extract spiked into peptides from human K562 cell lysates at three different ratios to simulate protein abundance change comparisons. SWATH-MS performance was assessed using local and external assay libraries of varying complexities and proteome compositions. These experiments demonstrated that local seed libraries integrated with external assay libraries achieve better performance than local assay libraries alone, in terms of the number of identified peptides and proteins and the specificity to detect differentially abundant proteins. Our findings show that the performance of extended assay libraries is influenced by the MS/MS feature similarity of the seed and external libraries, while statistical analysis using multiple testing corrections increases the statistical rigor needed when searching against large extended assay libraries. Data Independent Acquisition (DIA) 1 mass spectrometry workflows are gaining increasing use for proteomic analysis of model systems (1-8). The first integrated DIA and quantitative analysis protocol, termed SWATH (2) was shown to offer accurate, reproducible, and robust proteomic quantification (9 -14). DIA offers advantages over conventional IDA methods (15) by overcoming the stochastic, intensity-based selection of peptide precursors-a problem which typically leads to inconsistent peptide detection and quantitation between replicate runs. By overcoming this problem, DIA is highly suited for large-scale comparative analyses as gaps in data points between samples are mostly eliminated. These digital, extensive proteome maps can be repeatedly mined for quantitative data by extracting ion chromatograms of defined peptides postacquisition, and yields fewer quantitative missing (NA) values than IDA. An important concept in DIA analysis is use of a LC-retention time referenced spectral ion assay library to enable peptide identification from DIA generated multiplexed MS/MS spectra (10,13,16). The depth and quality of this spectral reference library directly correlates with experimental outcome, therefore we consider it is essential to explore and understand this variable in detail.The reference assay library should contain all the prior knowle...