Enzymatic digests, mapping

Peterson DS, Rohr T, Svec FK, Frechet JMJ (2003) Dual-function microanalyt-ical device by in situ photolithographic grafting of porous polymer monolith integrating solid-phase extraction and enzymatic digestion for peptide mass mapping. Anal Chem 75 5328... 

The easiest way to detect a protein modification seems to be the mass measurement of all peptides generated by enzymatic digestion. The comparison with the predicted peptide masses from the sequence of the protein identifies unmodified peptides and unexplained masses would give indications to modified peptides. Unfortunately, this is not a suitable approach in practice. In many peptide mapping experiments done with the MALDI mass mapping technique, up to 30% of the measured masses remain unexplained. This is probably due to protein contaminations from human keratins, chemical modifications introduced by gel electrophoresis and the digestion procedure, and other proteins present at low levels in the piece excised from the sodium dodecyl sulfate gel. The detection of a protein modification requires a more specific analysis. 

Figure 6.5 LC/MS/MS strategy for mapping glycoproteins and proteins via reduction, alkylation, and enzymatic digestion, followed by three LC/MS and LC/MS/MS experiments. (Reprinted with permission from Carr et al., 1993. Copyright 1993 Cold Spring Harbor Laboratory Press.)...

Figure 6.7 Comparative peptide map of enzymatic digests of bovine ribonuclease B. (A) TIC trace of digested RCM-glycoprotein with trypsin. (B) TIC trace of digested RCM-glycoprotein with trypsin and PNGase F. (Reprinted with permission from Liu et al., 1993. Copyright 1993 Elsevier.)...

Kassel, D. B. Shushan, B. Sakuma, T. Salzmann, J. P. 1994. Evaluation of packed capillary perfusion column HPLC/MS/MS for the rapid mapping and sequencing of enzymatic digests. Anal. Chem., 66,236-243. 

Tryptic Maps of Relaxin and Relaxin B-chain. Digestion of the A-chain of human relaxin with trypsin can theoretically result in the release of five fragments that of the B-chain in the release of six fragments as illustrated in Table II. A typical tryptic map of relaxin B-chain is shown in Figure 2. The peptide was reduced and carboxymethylated with iodoacetic acid before enzymatic digestion. The peptide assignments were made after analysis of the peaks by amino acid hydrolysis for amino acid composition and confirmed by fast atom bombardment mass spectrometry (FAB-MS) as shown in Table IH... 

FIGURE 6 Sketch showing the need for two enzymatic digestions, which contain different cleavage sites to enable the construction of the primary sequence from the sequenced peptide maps. 

Alkylammonium acetate buffer systems have also attracted limited attention for the separation of tryptic digests of proteins on octadecyl silica 60). The ammonium formate or trifluoroacetate buffers have also been used in the reversed-phase HPLC separation of the tryptic, chymotryptic, and carboxypeptidase A digests 136) of a-melanotropin and its N,0-diacetylserine analog as well as for enzymatic digests of the adrenocorticotropic family 137). Ammonium bicarbonate eluent systems have proved useful in mapping studies due to the eluent s volatility and differ-... 

The other isoforms, Iso-2 and Iso-3, expressed in the E. coli fermentation of IFN-a-2b, were characterized using a similar approach. The isolated Iso-2 and Iso-3 were enzymatically digested with trypsin, and the resulted peptide mixtures were mass-mapped using RP-HPLC/ESI-MS. The results indicated that Iso-2 was a correctly folded IFN-a-2b acetylated on the amino group of the N-terminal cysteine. Iso-3 was similarly determined to be a glutathionated form (Cys-98) of the partially reduced IFN-a-2b that was pyruvated on the N-terminal cysteine. The complete structures for IFN-a-2b, Iso-2, Iso-3, and Iso-4 are shown in Figure 19-11. 

An important development in high-throughput protein identifieation is the introduction of protein database searching [111]. After separation on ID- or 2D-GE, the proteins were blotted onto a membrane and enzymatically digested after reduction and alkylation. The tryptic peptide mixture is analysed by MALDl-MS to achieve a peptide map or peptide mass fingerprint (PMF). The m/z information of the peptides is used to search the protein database, e.g., the Protein Identification Resource (PIR) database [112-114]. If the mass of just 4-6 tryptic peptides is accurately measured (between 0.1 and 0.01%), a useful database search can be performed. 

Peterson, D.S. Rohr, T. Svec, F. Frechet, J.M.J. Dual-function Microanalyti-cal Device by In Situ Photolithographic Grafting of Porous Polymer Monolith Integrating Solid-Phase Extraction and Enzymatic Digestion for Peptide Mass Mapping, Anal. Chem. 75, 5328-5335 (2003). 

The chemical properties of the platinated DNA, termed M13-12A-Pt(-)-Stu I, were investigated by enzymatic, digestion and gel electrophoresis experiments. Platinum completely inhibits cleavage of the DNA by Stu I, as expected from the earlier restriction enzyme mapping studies. In addition, the cis-[Pt(NH3)2 d(pGpG) ] and cA-[Pt(NH3)2 d(pApG) ] intrastrand crosslinks were... 

The nuclear matrix. The lipid bilayers, the histones and other soluble proteins, and the DNA can all be removed from nuclei by exfracfion and enzymatic digestion. An insoluble residue, the nuclear matrix, is left. Largely protein in nature, this matrix is spread throughout the nucleus. Remnants of the membranes remain in the form of profeins that were in or along the bilayer. The nucleolus is clearly defined. The DNA appears to be bound to the nuclear matrix proteins. A specific 320-kb piece of a Drosophila chromosome has been mapped and used fo locate nontran-scribed scaffold (or mafrix) affachmenf regions of DNA bound to matrix proteins. These were found af intervals of 26-112 kb, fhe infervening loops confain-ing up to five or more A 120-kDa... 

---