12/18/2023 0 Comments Digested organics incubatorThe second aspect for adoption of MS sample preparation automation system are SRM assays of clinical value or the ability to monitor a broader number in a pseudo-discovery mode for research. Automation, and in this case using a 96 well format, can address many of these requirements 13. To enhance precision and accuracy, each sample preparation step must have accurate liquid transfers, be initiated and stopped at a consistent time, be performed at a controlled temperature and have good mixing for uniform reactions. It is, therefore, essential to have a highly controlled and standardized digestion method to meet the precision and reproducibility standards required for reliable biomarker verification. Optimal digestion conditions depend on both general and protein-specific factors including the trypsin-to-substrate ratio, buffer composition, protein structure, and the particular amino acid sequence and post-translational modifications adjacent to cleavage sites. Previous studies have shown that sample preparation, particularly the trypsin digestion step, is a major source for variability in LC-MS/MS analysis 10– 12. Sample preparation for LC-MS/MS analysis is a multi-step process involving i) protein solubilization and denaturation, ii) disulfide bond reduction and cysteine-blocking to ensure consistent cysteine masses, iii) digestion of proteins into peptides with a site-specific protease (most often trypsin), and iv) clean-up to remove salts, denaturing agents, and other interfering molecules (typically by solid-phase extraction) 8– 9. The development of a fast, highly reproducible, and completely hands-free MS protein sample preparation workflow would make the entire pathway from biomarker discovery to biomarker validation more robust. However, throughput, reproducibility, time, and cost remain longstanding barriers to large-scale MS sample processing 6– 7. Therefore, reproducible sample preparation is fundamental to success. Regardless of the MS approach, accuracy and precision of the quantitative measurements is critical. SRM is the method of choice for targeted biomarker validation because it enables specific, precise quantitation at higher throughput 3– 5. Once putative markers are identfied, targeted proteomic strategies such as selected reaction monitoring mass spectrometry (SRM) are typically used for verification and validation. Global protein discovery requires an in-depth quantitative analysis of the proteome using data-dependent (shotgun) 2 or data-independent acquisition (DIA) methods on 10s to 100s of samples. Quantitative LC/MS/MS experiments allow researchers to identify differences between the relative amounts of proteins in different sample populations, revealing biomarkers and providing insight into pathophysiology. These proteotypic peptides, as surrogates for the corresponding protein, are more easily measured using a range of liquid chromatography (LC) and mass spectrometry (MS) strategies. For proteomic analysis, proteins from complex samples derived from bodily fluids, biopsies, or cultured cells are typically digested into smaller peptides by protease digestion. Reproducible quantification of peptides from more than 70 plasma proteins was observed across replicates, days, instruments, and laboratory sites, demonstrating the broad applicability of this approach.īiomarker research and development consists of two phases: 1) global protein discovery to identify biomarker candidates and 2) verification and validation to confirm biomarker performance in a larger number of samples 1. An automated trypsin digestion workflow yields uniformly-processed samples in less than 5 hours. Similar results were obtained when the workflow was transferred to a second site: 93% of peptides had CVs below 20%. In a highly multiplexed SRM assay targeting more than 70 proteins, 90% of the transitions from 6 plasma samples repeated on 3 separate days had total CVs below 20%. In a selected reaction monitoring (SRM) assay targeting 6 plasma biomarkers and spiked β-galactosidase, mean intra-day and inter-day CVs for 5 serum and 5 plasma samples over 5 days were <20%. Peptide cleanup is accomplished by online diversion during the LC/MS/MS analysis. We established an automated sample preparation workflow with a total processing time for 96 samples of 5 hours, including a 2-hour incubation with trypsin. Scaling these procedures for the analysis of numerous complex biological samples can be tedious and time-consuming, as there are many liquid transfer steps and timed reactions where technical variations can be introduced and propagated. Sample preparation for protein quantification by mass spectrometry requires multiple processing steps including denaturation, reduction, alkylation, protease digestion, and peptide cleanup.
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