Identification strategies

Clauser, K. R. Baker, P Burlingame, A. L. Role of accurate mass measurement (+10ppm) in protein identification strategies employing MS or MS/MS and database searching. Anal. Chem. 1999,71,2871-2882. 

Protein Identification Strategies 15.2.8.1 Peptide Mass Fingerprinting (PMF)... 

FIGURE 2.1. Initial Theoretical Hazard Identification Strategy. 

The presence of gross errors invalidates the statistical basis of the common data reconciliation procedures, so they must be identified and removed. Gross error detection has received considerable attention in the past 20 years. Statistical tests in combination with an identification strategy have been used for this purpose. A good survey of the available methodologies can be found in Mah (1990) and Crowe (1996). 

Clauser, K.R. Baker, P. Burlingame, A. Role of Accurate Mass Measurement ( 10 Ppm) in Protein Identification Strategies Employing MS or MS/MS and Database Searching. Anal. Chem. 1999, 71, 2871-2882. 

The MS/MS identification strategy is based on the premise that much of the parent drug structure will be retained in the metabolites, impurities, or degradants (Perchalski et al., 1982 Lee et al.,... 

Screening large pools of compounds has several disadvantages, and often results in missed or false activities. To obviate such limitations, different pooling and structure identification strategies such as orthogonal libraries and binary encoded libraries have been developed and reported to provide examples of bioactive molecules. 

Figure 4.2 Schematic overview of two protein identification strategies commonly followed in proteomics. Protein samples are separated by either two-dimensional (2-D) or one-dimensional (1 -D) polyacrylamide gel electrophoresis (PAGE). In both strategic tracks, proteins are converted into a set of peptides by enzymatic digestion (e.g., with trypsin) prior to MS analysis. Peptide mass fingerprinting (PMF) by MALDl MS is predomi-...

Fig. 5.6 Possible identification strategy for nnknown migrants using GC-MS.

The bottom-up approach very much resembles classical protein identification strategies. The proteins in the proteome are first separated by 2D-GE (Ch. 17.3), or in some cases by SCX, size-exclusion (SEC), or affinity (AfC) chromatography. Specific proteins are excised from the gel, blotted, or electroeluted. The protein is digested, and the digest is analysed by LC-MS. The EC separation involves either RPLC with microcapillary or nano-LC columns (Ch. 17.5.2), or 2D-LC with typically SEC or SCX in the first dimension and RPLC in the second (Ch. 17.5.4). Alternatively, the sample may be introduced via either direct-infusion nano-ESl (Ch. 17.2), CE-MS (Ch. 17.5.6), or a microfluidic device coupled to MS (Ch. 17.5.5). 

While all these strategies rely on specific properties of phosphopeptides in MS analysis, a more global approach involving shotgun protein identification strategies and SEQUEST database searching (Ch. 18.3.2) is demonstrated by the group of Yates for protein complexes and lens tissue proteins [29]. 

In standard MS proteomics, well-established and efficient protocols allow for the en masse identification of proteins from tissue extracts. A key step within this process is the initial separation and purification of the molecules before submitting them to the mass spectrometer. However, in MSI studies, a general identification strategy for proteins, peptides, and metabolites is still missing due to several reasons. First, in comparison to LC-MS-based... 

Figure 4.2. Model identification strategy suggested by Ljung [171].

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