Proteins peptide workflows

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.

Of the fragmentation techniques MALDI-ISD, ECD, and ETD, it is only the latter that is directly compatible with the requirements and timescale of an online HX-MS workflow. By combining the classic bottom-up HX-MS workflow with gas-phase fragmentation by ETD, detailed information on protein HX can be obtained [47,57]. In such a combined workflow, enzymatic solution-phase cleavage is followed by automated (data-dependent acquisition) or manual (targeted) selection of peptides for gas-phase cleavage by ETD [57] (Eigure 8.6). 

Figure 8.6 The bottom-up HX-ETD workflow. HX, quench and solution-phase cleavage with enzymes are analogous to the classical bottom-up workflow (see Figure 1.2). Briefly, HX is initiated by dilution in Dfi. At several time points, aliquots are quenched to pH 2.5 and 0°C and digested with an acid-stable protease. The m/z values of the peptides are detected in MS survey scans followed by ETD gas-phase fragmentation. In top-down HX-ETD workflows, gas-phase fragmentation is performed on the intact protein (see Chapter 9). Figure adapted with permission from [41] 2014 American Chemical Society. (See insert for color representation of the figure.)...

In the online HX-MS/MS workflow, ETD fragmentation can be set up for all peptides in order to obtain as much site-specific HX information as possible, or alternatively, a targeted HX-ETD approach [47, 57] can be used to delineate site-specific HX only for selected peptides that display altered deuterium uptake upon ligand binding or across different protein states (Figure 8.9). 

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...

Figure 10.3 Typical mass spectral data obtained on a histidine-containing peptide generated in the experimental workflow shown in Figure 10.2. The data shown are from a histidine-containing peptide of sequence YTPHEETNNESF from the G protein-coupled receptor rhodopsin. The two mass spectra were obtained after the protein was exchanged for 0 and 72 h at pH 8.0. Reproduced with permission from Ref [26]. 2010, American Chemical Society...

Lopez, M.F., Mikulskis, A., Kuzdzal, S., et al (2007) A novel, high-throughput workflow for discovery and identification of serum carrier protein-bound peptide biomarker candidates in ovarian cancer samples. Clin. Chem., 53 (6), 1067-1074. 

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). 

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