A Sensitive and Controlled Data-Independent Acquisition Method for Proteomic Analysis of Cell Therapies.

Mass spectrometry (MS)-based proteomic measurements are uniquely poised to impact the development of cell and gene therapies. With the adoption of rigorous instrumental performance qualifications (PQs), large-scale proteomics can move from a research to a manufacturing control tool. Especially suited, data-independent acquisition (DIA) approaches have distinctive qualities to extend multiattribute method (MAM) principles to characterize the proteome of cell therapies. Here, we describe the development of a DIA method for the sensitive identification and quantification of proteins on a Q-TOF instrument. Using the improved acquisition parameters, we defined a control strategy and highlighted some metrics to improve the reproducibility of SWATH acquisition-based proteomic measurements. Finally, we applied the method to analyze the proteome of Jurkat cells that here serves as a model for human T-cells. Raw and processed data were deposited in PRIDE (PXD029780).

Combining quantitative and qualitative approaches using Sequential Window Acquisition of All Theoretical Fragment-Ion methodology for the detection of pharmaceuticals and related compounds in river fish extracted using a sample miniaturized method.

A fast method for analysis of 47 pharmaceuticals active compounds (PhACs) in fish muscle has been developed and validated addressing the parameters accuracy, precision (intraday and interday), matrix effect at three spiking levels: 5, 25 and 50 ng PhAC g-1 in fish as well as linearity, limit of detection (LOD), limit of quantification (LOQ). Sixteen protocols were performed varying extraction techniques, solvents, sample filtration and clean-up step. The selected method was based on an ultrasound extraction with acidified mixture of acetonitrile and isopropyl alcohol followed by a clean-up step using Z-Sep/C18 sorbents. Quantitative analysis of the PhACs in the extracts was accomplished by UPLC- QTOF-MS using Sequential Window Acquisition of All Theoretical Fragment-Ion, SWATHTM acquisition technology. 90% Of the compounds presented extraction recoveries between 60 and 130% with LOQ between 0.2 and 11 ng g-1. The validated method was applied to the analysis of 32 muscle samples from thirteen different species of fish collected in four European river basins (Adige, Evrotas, Llobregat and Sava). A total of ten compounds were found in fish samples with diltiazem as the most frequently detected one followed by carbamazepine and caffeine. Additionally, by taking advantage of the information-rich mass spectral data from the SWATH mode acquisition, the raw data were reprocessed for the presence of the most prescribed 250 pharmaceuticals, metabolites, and drugs of abuse previously reported to occur in the aquatic environment. By considering the mass errors of the molecular ion (˂± 3 ppm) and one characteristic fragment ion (˂±10 ppm) as well as the Library score and the Formula Finder score of the data processing software six compounds were retrieved, and eventually four of them confirmed with authentic standards: cocaine and its metabolite benzoylecgonine, the stimulant nicotine, and the antibiotic ofloxacin. Two lipid regulators, lovastatin and simvastatin, were determined as a false positive.

Development and validation of an analytical method for determination of pharmaceuticals in fish muscle based on QuEChERS extraction and SWATH acquisition using LC-QTOF-MS/MS system.

This study aimed at developing an analytical method for the extraction and quantification of 21 pharmaceutical actives compounds (PhACs) present in fish muscle. Using Norwegian Atlantic salmon as matrix, two extraction methods for PhACs were tested: ultrasound extraction (USE) using methanol (MeOH), acetonitrile (MeCN) or a mixture of MeCN:MeOH (1:1, v/v) as extracting solvents, and QuEChERS method using three different extraction salts. After selecting QuEChERS Original as extracting method of the analytes, three different clean-up methods were evaluated with respect to their efficiency to remove coextracted fat and lipids such as Enhanced Matrix Removal (EMR) and HLB prime. The dispersive-SPE EMR yielded the best recoveries for 21 of 27 analytes. PhACs were quantified by UPLC-MS/MS using SWATH acquisition mode. The method was validated in terms of recoveries, accuracy, linearity, precision, matrix effects at three levels of concentration: 25, 200 and 500 ng g-1 dw of fish muscle. For the majority of the analytes the recoveries were over 70%. Finally, the validated method was applied to natural riverine fish from the Evrotas river (Greece) and the Adige river (Italy) with positive findings for acetaminophen, propranolol, and venlafaxine reaching concentrations as high as 80 ng g-1 of muscle.

Generation of High-Quality SWATH® Acquisition Data for Label-free Quantitative Proteomics Studies Using TripleTOF® Mass Spectrometers.

Data-independent acquisition is a powerful mass spectrometry technique that enables comprehensive MS and MS/MS analysis of all detectable species, providing an information rich data file that can be mined deeply. Here, we describe how to acquire high-quality SWATH® Acquisition data to be used for large quantitative proteomic studies. We specifically focus on using variable sized Q1 windows for acquisition of MS/MS data for generating higher specificity quantitative data.

Automated SWATH Data Analysis Using Targeted Extraction of Ion Chromatograms.

Targeted mass spectrometry comprises a set of methods able to quantify protein analytes in complex mixtures with high accuracy and sensitivity. These methods, e.g., Selected Reaction Monitoring (SRM) and SWATH MS, use specific mass spectrometric coordinates (assays) for reproducible detection and quantification of proteins. In this protocol, we describe how to analyze, in a targeted manner, data from a SWATH MS experiment aimed at monitoring thousands of proteins reproducibly over many samples. We present a standard SWATH MS analysis workflow, including manual data analysis for quality control (based on Skyline) as well as automated data analysis with appropriate control of error rates (based on the OpenSWATH workflow). We also discuss considerations to ensure maximal coverage, reproducibility, and quantitative accuracy.

Proteomic profiling of Bacillus licheniformis reveals a stress response mechanism in the synthesis of extracellular polymeric flocculants.

Some bioflocculants composed of extracellular polymeric substances are produced under peculiar conditions. Bacillus licheniformis CGMCC2876 is a microorganism that secretes both extracellular polysaccharides (EPS) and poly-gamma-glutamic acid (γ-PGA) under stress conditions. In this work, SWATH acquisition LC-MS/MS method was adopted for differential proteomic analysis of B. licheniformis, aiming at determining the bacterial stress mechanism. Compared with LB culture, 190 differentially expressed proteins were identified in B. licheniformis CGMCC2876 cultivated in EPS culture, including 117 up-regulated and 73 down-regulated proteins. In γ-PGA culture, 151 differentially expressed proteins, 89 up-regulated and 62 down-regulated, were found in the cells. Up-regulated proteins involved in amino acid biosynthesis were found to account for 43% and 41% of the proteomes in EPS and γ-PGA cultivated cells, respectively. Additionally, a series of proteins associated with amino acid degradation were found to be repressed under EPS and γ-PGA culture conditions. Transcriptional profiling via the qPCR detection of selected genes verified the proteomic analysis. Analysis of free amino acids in the bacterial cells further suggested the presence of amino acid starvation conditions. EPS or γ-PGA was synthesized to alleviate the effect of amino acid limitation in B. licheniformis. This study identified a stress response mechanism in the synthesis of macromolecules in B. licheniformis, providing potential culture strategies to improve the production of two promising bioflocculants.