Tryptic fragments, extracting

In addition to the attempts at unraveling the modification site, Dive and coworkers also constructed two biotinylated A/BPs, 22 and 23 (Scheme 3c), and used these to study the difference in affinity- and photoaffinity MMP enrichment from a complex proteome [57]. For this, tumor extracts were spiked with hMMP-12 and hMMP-8, after which compounds 22 and 23 were applied, followed by streptavidin-coated magnetic beads for MMP pull-down. Affinity-based labeling with 23 appeared superior to photoaffinity-based labeling with 22 in terms of quantity of captured MMPs and identification of the tryptic fragments by mass spectrometry. 

The study of protein structure, function, quantity, and interactions during maturation and progression of disease is referred to as proteomics. Analytical approaches that use a combination of two-dimensional (2-D) gel electrophoresis for protein separation and MS analysis for protein identification followed by database searches is a widely practiced proteomics strategy.The tryptic peptides extracted from gels are analyzed by MALDI-TOF MS and microcolunm or capillary LC tandem mass spectrometry (MS/MS) techniques. Typically, the MALDI-TOF MS techniques are used to quickly identify peptide fragments and confirm the presence of known proteins. Nano-scale capillary LC/MS/MS techniques (using 50-100 pm diameter columns, operating at flow rates of 20-500 nL/min) are... 

Peptides extracted from casein with N, N-dimethyl formamide have complex electrophoretic patterns identical to those of the fraction first prepared by Long and co-workers and called X-casein (El-Negoumy 1973). These peptides are identical electrophoretically to those released by the action of plasmin, which is present in fresh raw milk, upon asr casein (Aimutis and Eigel 1982). Two of these peptides have tryptic peptide maps and molecular weights identical to those of a pair of the peptides produced by plasmin degradation of asl-casein. These peptides appear to be fragments of a8l-casein which are present in milk as the result of plasmin proteolysis. More definitive information on their primary structure is needed before nomenclature for these fragments can be established. 

In the epitope extraction experiment, cAPP(724-770) was cleaved by trypsin in solution. Three proteolytic fragments were identified by MALDI-FTICR-MS, APP(727-747), APP(752-763) and APP(764-770) (Figure 4A). The same fragments were also detected in the supernatant fraction after the affinity column was incubated with the tryptic peptide mixture. After washing with 30 ml PBS buffer, no fragments were detected in the last 1 ml of the washing fraction (data not shown). In the elution... 

A ricin detection method has been developed that couples LC-MS/MS with an enzyme-linked immunosorbent assay (ELISA).The analyte target for MS detection is adenine, which is released from ricin during the assay. Adenine detection limits were achieved at 0.1 ng/mL or 1.56 pM in a 500-pL sample volume with this method. This LC-MS method may be chosen over other techniques because milk and drinking water are the intended real-world application for this protocol [77]. Another MS/MS technique detects tryptic peptide fragments from ricin produced by an immunoassay technique. This technique utilizes the inherent speed of MS analysis to confirm peptide identity. This technique is applied to the detection of ricin in food and body fluids such as blood serum and saliva [78]. A similar LC-MS/MS method uses an organic solvent-assisted tryptic digest to prepare samples of crude ricin extracts. This sample preparation... 

Figure 11.32 Mass spectra for the analysis of tryptic digest of HRP. (a) Direct MS analysis (sample tryptic digest of 1 mg mL HRP diluted tenfold with 100 mM HAc) (h) MS analysis after extraction. The peaks of gly-copeptides or their fragments are marked.

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