Workflow spectrometry

FIGURE 1 Example of a gel-free-oriented proteomics nano-LC/MS-MS workflow in which bacterial culture proteins digested to tryptic peptides are separated via LC and peptides subsequently analyzed by mass spectrometry. In the process, the spectrometer rapidly cycles every few seconds and examines a size window in which peptide-derived MSI ions are analyzed to define MS/MS (MS2) spectra. The MS/MS (MS2) spectrum generated for each peptide then enters a bioinformatic pipeline for sequence identification, statistical validation, and quantification. 

Figure 2. Workflow of an LC-MS/MS experiment. A mixture of peptides from a protein sample digest is separated by reversed-phase chromatography on a nano-flow HPLC. The peptides elute from the RP column and are ionized by an electrospray source. In the first stage of mass spectrometry, m/z values and charge states for each precursor ion are determined and the most abundant precursor ions are selected for analysis in the second stage. The ions are then fragmented with by collision-induced dissociation (CID) a gas to produce fragment ions which are detected. Using the mass (from MS-1) and sequence information (from MS-2) protein sequence databases are searched to provide peptide identifications and protein matches.

In a standard hydrogen exchange experiment (see Section 2.3.4 for variations in experimental workflow), the sample is deuterated for a predetermined time (milliseconds and longer times), and the reaction is slowed down several orders of magnitude by the addition of an acidic quench solution. Quenched reactions can be subjected to direct mass spectrometry analysis or immediately flash frozen in liquid nitrogen and stored at -80°C in multiple aliquots for future analysis. These flash-frozen samples need to be thawed prior to mass spectrometry. It is important to note that freeze-thaw cycles contribute to signal loss due to back-exchange (this is described further in Section 2.3.8), which... 

Figure 10.2 Schematic representation of the experimental workflow used in mass spectrometry-based experiments to determine the pK of histidine residues in proteins. The first step of the protocol involves incubation of the protein in a series of D,0 buffers at different pH values for at least 2 days before the hydrogen exchange reactions are quenched. The protein samples from each hydrogen exchange reaction are then digested with proteolytic enzyme(s), and the proteolytic fragments are analyzed by LC-TSI-MS to determine the deuterium content cjf histidine-ccjntaining peptides...

So, what criteria will emerge as the most desirable analytical figure of merit for high-performance LC/MS analysis in drug metabolism It is our sincere hope that this book will provide an updated perspective on mass spectrometry in drug metabolism and disposition with recent applications, novel technologies, and innovative workflows. 

Briefly, the workflow for these determinations is as follows preparation of a protein sample, parallel treatments of reduced and unreduced samples with iodoacetamide, digestion of proteins with trypsin, and analysis of peptides by Liquid Chromatography coupled with Mass Spectrometry (LC-MS). 

Li, A.C., Ding, J., Jiang, X., et al. (2009) Two-injection workflow for a liquid chromatography/LTQ-Orbitrap system to complete in vivo biotransformation characterization demonstration with buspirone metabohte identification. Rapid Communications in Mass Spectrometry, 23,3003-3012. 

Geddes, K., Adamson, G., Dube, N., Crathern, S., King, R.C. (2009) Semi-automated tandem mass spectrometric (MS/MS) triple quadrupole operating parameter optimization for high-throughput MS/MS detection workflows. Rapid Communications in Mass Spectrometry, 23(9), 1303-1312. 

Luzzatto-Knaan T, Melnik AV, Dorrestein PC. Mass spectrometry tools and workflows for revealing microbial chemistry. Analyst 2015 140 4949-66. 

Fig. 3.1 Workflow of sample preparation approaches for microbial analysis. MS mass spectrometry...

Gundry RL, White MY, Murray Cl, et al. Preparation of proteins and peptides for mass spectrometry analysis in a bottom-up proteomics workflow. Curr Protoc Mol Biol. 2009 88 10.25.1-10.25.23. doi 10.1002/0471142727.mbl025s88. 

Vu TN, Valkenborg D, Smets K, Verwaest KA, Dommisse R, Lemiere F, et al. An integrated workflow fra" robust alignment and simplified quantitative analysis of NMR spectrometry data. BMC Bioinformatics 2011 12 405. 

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