High-throughput proteomics of nanogram-scale samples with Zeno SWATH MS.

The possibility to record proteomes in high throughput and at high quality has opened new avenues for biomedical research, drug discovery, systems biology, and clinical translation. However, high-throughput proteomic experiments often require high sample amounts and can be less sensitive compared to conventional proteomic experiments. Here, we introduce and benchmark Zeno SWATH MS, a data-independent acquisition technique that employs a linear ion trap pulsing (Zeno trap pulsing) to increase the sensitivity in high-throughput proteomic experiments. We demonstrate that when combined with fast micro- or analytical flow-rate chromatography, Zeno SWATH MS increases protein identification with low sample amounts. For instance, using 20 min micro-flow-rate chromatography, Zeno SWATH MS identified more than 5000 proteins consistently, and with a coefficient of variation of 6%, from a 62.5 ng load of human cell line tryptic digest. Using 5 min analytical flow-rate chromatography (800 µl/min), Zeno SWATH MS identified 4907 proteins from a triplicate injection of 2 µg of a human cell lysate, or more than 3000 proteins from a 250 ng tryptic digest. Zeno SWATH MS hence facilitates sensitive high-throughput proteomic experiments with low sample amounts, mitigating the current bottlenecks of high-throughput proteomics.

Optimization of Microflow LC Coupled with Scanning SWATH and Its Application in Hepatocellular Carcinoma Tissues.

Scanning SWATH coupled with normal-flow LC has been recently introduced for high-content, high-throughput proteomics analysis, which requires a relatively large amount of sample injection. Here we established the microflow LC coupled with Scanning SWATH for samples with relatively small quantities. First, we optimized several key parameters of the LC and MS settings, including C18 particle size for the analytical column, LC gradient and flow rate, as well as effective ion accumulation time and isolation window width for MS acquisition. We then compared the optimized Scanning SWATH method with the conventional variable window SWATH (referred to as SWATH) method. Results showed that the total ion chromatogram signals in Scanning SWATH were 10 times higher than that of SWATH, and Scanning SWATH identified 12.2-22.2% more peptides than SWATH. Finally, we employed 120 min Scanning SWATH to acquire the proteomes of 62 formalin-fixed, paraffin-embedded (FFPE) tissue samples from 31 patients with hepatocellular carcinoma (HCC). Altogether, 92 334 peptides and 8516 proteins were quantified. Besides the reported biomarkers, including ANXA2, MCM7, SUOX, and AKR1B10, we identified new potential HCC biomarkers such as CST5, TP53, CEBPB, and E2F4. Taken together, we present an optimal workflow integrating microflow LC and Scanning SWATH that effectively improves the protein identification and quantitation.

A comprehensive LFQ benchmark dataset on modern day acquisition strategies in proteomics.

In the last decade, a revolution in liquid chromatography-mass spectrometry (LC-MS) based proteomics was unfolded with the introduction of dozens of novel instruments that incorporate additional data dimensions through innovative acquisition methodologies, in turn inspiring specialized data analysis pipelines. Simultaneously, a growing number of proteomics datasets have been made publicly available through data repositories such as ProteomeXchange, Zenodo and Skyline Panorama. However, developing algorithms to mine this data and assessing the performance on different platforms is currently hampered by the lack of a single benchmark experimental design. Therefore, we acquired a hybrid proteome mixture on different instrument platforms and in all currently available families of data acquisition. Here, we present a comprehensive Data-Dependent and Data-Independent Acquisition (DDA/DIA) dataset acquired using several of the most commonly used current day instrumental platforms. The dataset consists of over 700 LC-MS runs, including adequate replicates allowing robust statistics and covering over nearly 10 different data formats, including scanning quadrupole and ion mobility enabled acquisitions. Datasets are available via ProteomeXchange (PXD028735).

Ultra-fast proteomics with Scanning SWATH.

Accurate quantification of the proteome remains challenging for large sample series and longitudinal experiments. We report a data-independent acquisition method, Scanning SWATH, that accelerates mass spectrometric (MS) duty cycles, yielding quantitative proteomes in combination with short gradients and high-flow (800 µl min-1) chromatography. Exploiting a continuous movement of the precursor isolation window to assign precursor masses to tandem mass spectrometry (MS/MS) fragment traces, Scanning SWATH increases precursor identifications by ~70% compared to conventional data-independent acquisition (DIA) methods on 0.5-5-min chromatographic gradients. We demonstrate the application of ultra-fast proteomics in drug mode-of-action screening and plasma proteomics. Scanning SWATH proteomes capture the mode of action of fungistatic azoles and statins. Moreover, we confirm 43 and identify 11 new plasma proteome biomarkers of COVID-19 severity, advancing patient classification and biomarker discovery. Thus, our results demonstrate a substantial acceleration and increased depth in fast proteomic experiments that facilitate proteomic drug screens and clinical studies.

A comprehensive CHO SWATH-MS spectral library for robust quantitative profiling of 10,000 proteins.

Sequential window acquisition of all theoretical fragment-ion spectra (SWATH) is a data-independent acquisition (DIA) strategy that requires a specific spectral library to generate unbiased and consistent quantitative data matrices of all peptides. SWATH-MS is a promising approach for in-depth proteomic profiling of Chinese hamster Ovary (CHO) cell lines, improving mechanistic understanding of process optimization, and real-time monitoring of process parameters in biologics R&D and manufacturing. However, no spectral library for CHO cells is publicly available. Here we present a comprehensive CHO global spectral library to measure the abundance of more than 10,000 proteins consisting of 199,102 identified peptides from a CHO-K1 cell proteome. The robustness, accuracy and consistency of the spectral library were validated for high confidence in protein identification and reproducible quantification in different CHO-derived cell lines, instrumental setups and downstream processing samples. The availability of a comprehensive SWATH CHO global spectral library will facilitate detailed characterization of upstream and downstream processes, as well as quality by design (QbD) in biomanufacturing. The data have been deposited to ProteomeXchange (PXD016047).

MetaboKit: a comprehensive data extraction tool for untargeted metabolomics.

We have developed MetaboKit, a comprehensive software package for compound identification and relative quantification in mass spectrometry-based untargeted metabolomics analysis. In data dependent acquisition (DDA) analysis, MetaboKit constructs a customized spectral library with compound identities from reference spectral libraries, adducts, dimers, in-source fragments (ISF), MS/MS fragmentation spectra, and more importantly the retention time information unique to the chromatography system used in the experiment. Using the customized library, the software performs targeted peak integration for precursor ions in DDA analysis and for precursor and product ions in data independent acquisition (DIA) analysis. With its stringent identification algorithm requiring matches by both MS and MS/MS data, MetaboKit provides identification results with significantly greater specificity than the competing software packages without loss in sensitivity. The proposed MS/MS-based screening of ISFs also reduces the chance of unverifiable identification of ISFs considerably. MetaboKit's quantification module produced peak area values highly correlated with known concentrations in a DIA analysis of the metabolite standards at both MS1 and MS2 levels. Moreover, the analysis of Cdk1Liv-/- mouse livers showed that MetaboKit can identify a wide range of lipid species and their ISFs, and quantitatively reconstitute the well-characterized fatty liver phenotype in these mice. In DIA data, the MS1-level and MS2-level peak area data produced similar fold change estimates in the differential abundance analysis, and the MS2-level peak area data allowed for quantitative comparisons in compounds whose precursor ion chromatogram was too noisy for peak integration.

Tracking genome-editing and associated molecular perturbations by SWATH mass spectrometry.

Advances in gene editing now allow reverse genetics to be applied to a broad range of biological systems. Ultimately, any modification to coding sequences requires confirmation at the protein level, although immunoblotting is often hampered by antibody quality or availability especially in non-model species. Sequential Window Acquisition of All Theoretical Spectra (SWATH), a mass spectrometry (MS) technology with exceptional quantitative reproducibility and accuracy, offers an ideal alternative for protein-based confirmation. Here, using genome edits in mouse, zebrafish and Bicyclus anynana butterflies produced using either homologous recombination or targeted nucleases, we demonstrate absence of the targeted proteins using SWATH, thus confirming successful editing. We show that SWATH is a robust antibody-independent alternative for monitoring gene editing at the protein level and broadly applicable across diverse organisms and targeted genome manipulation techniques. Moreover, SWATH concomitantly defines the global proteome response in the edited organism, which may provide pertinent biological insights.

Expanding the Use of Spectral Libraries in Proteomics.

The 2017 Dagstuhl Seminar on Computational Proteomics provided an opportunity for a broad discussion on the current state and future directions of the generation and use of peptide tandem mass spectrometry spectral libraries. Their use in proteomics is growing slowly, but there are multiple challenges in the field that must be addressed to further increase the adoption of spectral libraries and related techniques. The primary bottlenecks are the paucity of high quality and comprehensive libraries and the general difficulty of adopting spectral library searching into existing workflows. There are several existing spectral library formats, but none captures a satisfactory level of metadata; therefore, a logical next improvement is to design a more advanced, Proteomics Standards Initiative-approved spectral library format that can encode all of the desired metadata. The group discussed a series of metadata requirements organized into three designations of completeness or quality, tentatively dubbed bronze, silver, and gold. The metadata can be organized at four different levels of granularity: at the collection (library) level, at the individual entry (peptide ion) level, at the peak (fragment ion) level, and at the peak annotation level. Strategies for encoding mass modifications in a consistent manner and the requirement for encoding high-quality and commonly seen but as-yet-unidentified spectra were discussed. The group also discussed related topics, including strategies for comparing two spectra, techniques for generating representative spectra for a library, approaches for selection of optimal signature ions for targeted workflows, and issues surrounding the merging of two or more libraries into one. We present here a review of this field and the challenges that the community must address in order to accelerate the adoption of spectral libraries in routine analysis of proteomics datasets.

Statistical control of peptide and protein error rates in large-scale targeted data-independent acquisition analyses.

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) 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, as exemplified by the technique SWATH-MS, has emerged as a scalable method to achieve deep and consistent proteome coverage across large-scale data sets. We demonstrate that statistical concepts developed for discovery proteomics based on spectrum-centric scoring can be adapted to large-scale DIA experiments that have been analyzed with peptide-centric scoring strategies, and we 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 the accumulation of false positives across large-scale data sets. Furthermore, to increase the quality and reproducibility of published proteomic results, well-established confidence criteria should be reported for the detected peptide queries, peptides and inferred proteins.

SWATH-ID: An instrument method which combines identification and quantification in a single analysis.

Data-independent acquisition (DIA) approaches, such as SWATH® -MS, are showing great potential to reliably quantify significant numbers of peptides and proteins in an unbiased manner. These developments have enhanced interest in developing a single DIA method that integrates qualitative and quantitative analysis, eliminating the need of a prebuilt library of peptide spectra, which are created through data-dependent acquisition methods or from public repositories. Here, we introduce a new DIA approach, referred to as "SWATH-ID," which was developed to allow peptide identification as well as quantitation. The SWATH-ID method is composed of small Q1 windows, achieving better selectivity and thus significantly improving high-confidence peptide extractions from data files. Furthermore, the SWATH-ID approach transmits precursor ions without fragmentation as well as their fragments within the same SWATH acquisition period. This provides a single scan that includes all precursor ions within the isolation window as well as a record of all of their fragment ions, substantially negating the need for a survey scan. In this way all precursors present in a small Q1 window are associated with their fragment ions, improving the identification specificity and providing a more comprehensive and in-depth view of protein and peptide species in complex samples.

A multicenter study benchmarks software tools for label-free proteome quantification.

Consistent and accurate quantification of proteins by mass spectrometry (MS)-based proteomics depends on the performance of instruments, acquisition methods and data analysis software. In collaboration with the software developers, we evaluated OpenSWATH, SWATH 2.0, Skyline, Spectronaut and DIA-Umpire, five of the most widely used software methods for processing data from sequential window acquisition of all theoretical fragment-ion spectra (SWATH)-MS, which uses data-independent acquisition (DIA) for label-free protein quantification. We analyzed high-complexity test data sets from hybrid proteome samples of defined quantitative composition acquired on two different MS instruments using different SWATH isolation-window setups. For consistent evaluation, we developed LFQbench, an R package, to calculate metrics of precision and accuracy in label-free quantitative MS and report the identification performance, robustness and specificity of each software tool. Our reference data sets enabled developers to improve their software tools. After optimization, all tools provided highly convergent identification and reliable quantification performance, underscoring their robustness for label-free quantitative proteomics.

A randomised clinical trial of the effectiveness of home-based health care with telemonitoring in patients with COPD.

We studied the effect of telemonitoring in addition to usual care compared to usual care alone in patients with chronic obstructive pulmonary disease (COPD). A total of 110 patients with moderate to severe COPD were recruited from a specialist respiratory service in Northern Ireland. Patients had at least two of: emergency department admissions, hospital admissions or emergency general practitioner (GP) contacts in the 12 months before the study. Exclusion criteria were patients who had any respiratory disorder other than COPD, or were cognitively unable to learn the process of monitoring. Patients were randomised to receive six months of home telemonitoring with usual care, or six months of usual care. The primary outcome measure was disease-specific quality of life, as measured by the St George's Respiratory Questionnaire for COPD patients (SGRQ-C). Of 100 patients completing the study, 48 patients were randomised to telemonitoring and 52 patients were randomised to the control group. The SGRQ-C scores improved significantly in the intervention group compared to usual care (P = 0.001). The HADS anxiety score was significantly higher in the telehealth group compared to the usual care group (P = 0.01). There were significantly more contacts with the Community Respiratory Team in the telemonitoring group compared to the control group (P = 0.029). There were no significant between group differences in EQ-5D scores, HADS depression scores, GP activity, emergency department visits, hospital admissions or exacerbations. The total cost to the health service of the intervention over the 6-month study period was £2039, giving an estimated ICER of £203,900. In selected patients with COPD, telemonitoring was effective in improving health-related quality of life and anxiety, but was not a cost-effective intervention.

Albumin decrease is associated with spontaneous preterm delivery within 48 h in women with threatened preterm labor.

Threatened preterm labor (TPTL) accounts for ∼30% of pregnancy-related hospital admissions. Maternal peripheral leukocytes can be used to monitor a variety of physiological processes occurring in the body. Two high-throughput mass spectrometry methodologies, SWATH and iTRAQ, were used to study differentially expressed peripheral blood leukocyte lysate proteins in symptomatic women admitted for TPTL who had a preterm birth within 48 h (n = 16) and those who did not (n = 24). The SWATH spectral library consisted of 783 proteins. SWATH methodology quantified 258 proteins (using ≥2 peptides) and 5 proteins (ALBU, ANXA6, HNRPK, HSP90A, and PDIA1) were differentially expressed (p < 0.05, Mann-Whitney U). iTRAQ workflow identified 765 proteins; 354 proteins were quantified and 14 proteins (MIF, UBIQ, HXK3, ALBU, HNRPD, ST1A2, RS15A, RAP1B, CAN1, IQGA2, ST1A1, COX5A, ADDA, and UBQL1) were significantly different between the two groups of women (p < 0.05, Mann-Whitney U). Albumin was the only common differentially expressed protein in both SWATH (28% decrease) and iTRAQ studies (45% decrease). This decrease in albumin was validated using ELISA (11% decrease, p < 0.05, Mann-Whitney U) in another 23 TPTL women. This work suggests that albumin is a broad indicator of leukocyte activation with impending preterm birth and provides new future work directions to understand the pathophysiology of TPTL.

Development of a highly automated and multiplexed targeted proteome pipeline and assay for 112 rat brain synaptic proteins.

We present a comprehensive workflow for large scale (>1000 transitions/run) label-free LC-MRM proteome assays. Innovations include automated MRM transition selection, intelligent retention time scheduling that improves S/N by twofold, and automatic peak modeling. Improvements to data analysis include a novel Q/C metric, normalized group area ratio, MLR normalization, weighted regression analysis, and data dissemination through the Yale protein expression database. As a proof of principle we developed a robust 90 min LC-MRM assay for mouse/rat postsynaptic density fractions which resulted in the routine quantification of 337 peptides from 112 proteins based on 15 observations per protein. Parallel analyses with stable isotope dilution peptide standards (SIS), demonstrate very high correlation in retention time (1.0) and protein fold change (0.94) between the label-free and SIS analyses. Overall, our method achieved a technical CV of 11.4% with >97.5% of the 1697 transitions being quantified without user intervention, resulting in a highly efficient, robust, and single injection LC-MRM assay.

A repository of assays to quantify 10,000 human proteins by SWATH-MS.

Mass spectrometry is the method of choice for deep and reliable exploration of the (human) proteome. Targeted mass spectrometry reliably detects and quantifies pre-determined sets of proteins in a complex biological matrix and is used in studies that rely on the quantitatively accurate and reproducible measurement of proteins across multiple samples. It requires the one-time, a priori generation of a specific measurement assay for each targeted protein. SWATH-MS is a mass spectrometric method that combines data-independent acquisition (DIA) and targeted data analysis and vastly extends the throughput of proteins that can be targeted in a sample compared to selected reaction monitoring (SRM). Here we present a compendium of highly specific assays covering more than 10,000 human proteins and enabling their targeted analysis in SWATH-MS datasets acquired from research or clinical specimens. This resource supports the confident detection and quantification of 50.9% of all human proteins annotated by UniProtKB/Swiss-Prot and is therefore expected to find wide application in basic and clinical research. Data are available via ProteomeXchange (PXD000953-954) and SWATHAtlas (SAL00016-35).

Mapping differential interactomes by affinity purification coupled with data-independent mass spectrometry acquisition.

Characterizing changes in protein-protein interactions associated with sequence variants (e.g., disease-associated mutations or splice forms) or following exposure to drugs, growth factors or hormones is critical to understanding how protein complexes are built, localized and regulated. Affinity purification (AP) coupled with mass spectrometry permits the analysis of protein interactions under near-physiological conditions, yet monitoring interaction changes requires the development of a robust and sensitive quantitative approach, especially for large-scale studies in which cost and time are major considerations. We have coupled AP to data-independent mass spectrometric acquisition (sequential window acquisition of all theoretical spectra, SWATH) and implemented an automated data extraction and statistical analysis pipeline to score modulated interactions. We used AP-SWATH to characterize changes in protein-protein interactions imparted by the HSP90 inhibitor NVP-AUY922 or melanoma-associated mutations in the human kinase CDK4. We show that AP-SWATH is a robust label-free approach to characterize such changes and propose a scalable pipeline for systems biology studies.

Temporal regulation of EGF signalling networks by the scaffold protein Shc1.

Cell-surface receptors frequently use scaffold proteins to recruit cytoplasmic targets, but the rationale for this is uncertain. Activated receptor tyrosine kinases, for example, engage scaffolds such as Shc1 that contain phosphotyrosine (pTyr)-binding (PTB) domains. Using quantitative mass spectrometry, here we show that mammalian Shc1 responds to epidermal growth factor (EGF) stimulation through multiple waves of distinct phosphorylation events and protein interactions. After stimulation, Shc1 rapidly binds a group of proteins that activate pro-mitogenic or survival pathways dependent on recruitment of the Grb2 adaptor to Shc1 pTyr sites. Akt-mediated feedback phosphorylation of Shc1 Ser 29 then recruits the Ptpn12 tyrosine phosphatase. This is followed by a sub-network of proteins involved in cytoskeletal reorganization, trafficking and signal termination that binds Shc1 with delayed kinetics, largely through the SgK269 pseudokinase/adaptor protein. Ptpn12 acts as a switch to convert Shc1 from pTyr/Grb2-based signalling to SgK269-mediated pathways that regulate cell invasion and morphogenesis. The Shc1 scaffold therefore directs the temporal flow of signalling information after EGF stimulation.

Label-free quantitative proteomics trends for protein-protein interactions.

Understanding protein interactions within the complexity of a living cell is challenging, but techniques coupling affinity purification and mass spectrometry have enabled important progress to be made in the past 15 years. As identification of protein-protein interactions is becoming easier, the quantification of the interaction dynamics is the next frontier. Several quantitative mass spectrometric approaches have been developed to address this issue that vary in their strengths and weaknesses. While isotopic labeling approaches continue to contribute to the identification of regulated interactions, techniques that do not require labeling are becoming increasingly used in the field. Here, we describe the major types of label-free quantification used in interaction proteomics, and discuss the relative merits of data dependent and data independent acquisition approaches in label-free quantification. This article is part of a Special Issue entitled: From protein structures to clinical applications.

Pyruvate kinase is a dosage-dependent regulator of cellular amino acid homeostasis.

The glycolytic enzyme pyruvate kinase (PK) is required for cancer development, and has been implicated in the metabolic transition from oxidative to fermentative metabolism, the Warburg effect. However, the global metabolic response that follows changes in PK activity is not yet fully understood. Using shotgun proteomics, we identified 31 yeast proteins that were regulated in a PK-dependent manner. Selective reaction monitoring confirmed that their expression was dependent on PK isoform, level and activity. Most of the PK targets were amino acid metabolizing enzymes or factors of protein translation, indicating that PK plays a global regulatory role in biosynthethic amino acid metabolism. Indeed, we found strongly altered amino acid profiles when PK levels were changed. Low PK levels increased the cellular glutamine and glutamate concentrations, but decreased the levels of seven amino acids including serine and histidine. To test for evolutionary conservation of this PK function, we quantified orthologues of the identified PK targets in thyroid follicular adenoma, a tumor characterized by high PK levels and low respiratory activity. Aminopeptidase AAP-1 and serine hydroxymethyltransferase SHMT1 both showed PKM2- concentration dependence, and were upregulated in the tumor. Thus, PK expression levels and activity were important for maintaining cellular amino acid homeostasis. Mediating between energy production, ROS clearance and amino acid biosynthesis, PK thus plays a central regulatory role in the metabolism of proliferating cells.