Mass spectrometry peptide sequencing with

However, interpretation of, or even obtaining, the mass spectrum of a peptide can be difficult, and many techniques have been introduced to overcome such difficulties. These techniques include modifying the side chains in the peptide and protecting the N- and C-terminals by special groups. Despite many advances made by these approaches, it is not always easy to read the sequence from the mass spectrum because some amide bond cleavages are less easy than others and give little information. To overcome this problem, tandem mass spectrometry has been applied to this dry approach to peptide sequencing with considerable success. Further, electrospray ionization has been used to determine the molecular masses of proteins and peptides with unprecedented accuracy. 

Ermer, for example, utilized LCQ to monitor impurity profiles of various batches of ramorelix used in toxicological studies, clinical stndies and scale-up. Ramorelix is a synthetic glycosylated decapeptide with monoisotopic of 1530.7. The toxicological batch served as the benchmark against which all other batches were compared. Molecnlar weights of impurities were determined by ESI mass spectrometry, and nsed in conjunction with UV peak area % to gauge impurities in batches nsed in clinical trials. These impurity profiles were compared to those of batches used in the toxicologically qualified batch. Eour impurities were detected with the same value. They were believed to be diastereoisomers of ramorelix, i.e., a peptide sequence with one of the amino acids in the opposite enantiomeric form. 

Figure 2. Peptide maps (A-C) and MALDI-TOF mass spectra (D-F) of PVDF-bound transferrin (53 pmol) digested with trypsin in the presence of 50 pi of 1% RTX-100/10% acetonitrile/100 mM Tris, pH 8.0 (A,D), 1% octylglucopyranoside/10% acetonitrile/100 mM Tris, pH 8.0 (B,E), and 1% decylglucopyranoside/10% acetonitrile/100 mM Tris, pH 8.0 (CJF) as described in Materials and Methods. Ninety percent of the digestion was analyzed by HPLC ( 29 pmol based on Table I) and 0.5% ( 150 fmol) was used for MALDI-TOF mass spectrometry. Peptides 1 and 2 in A-C were amino terminally sequenced (Table II) and analyzed by MALDI-TOF mass spectrometry (Figure 3).

DR Owens, B Bothner, O Phung, K Harris, G Siuzdak. Aspects of oligonucleotide and peptide sequencing with MALDI and electrospray mass spectrometry. BioorgMed Chem 6(9) 1547—1554, 1998. 

Tandem mass spectrometry can be applied for analysis of peptide mixtures. The first quadrupole only passes one specific peptide ion, which is then fragmented in the collision chamber, i.e. amino acids are cleaved from the peptide chain. In the third quadrupole, the difference between mass peaks gives information about the amino acid sequence in the peptide. An example of peptide sequencing with ESI-MS/MS is shown in Fig. 4.20. Oligonucleotides and oligosaccharides can be analysed in a similar fashion. 

Other methods for determining the sequence of a polypeptide include mass spectrometry and comparison of partial peptide sequences with databases of known complete sequences. 

Mass spectrometry-based protein identification protocols have propelled proteomics to the forefront of biomedical research. Provided with available genomic and/or protein sequence information in databases, protein identification by MALDl-based peptide mass fingerprint mapping and LC-MS/MS and MALDl-TOF/TOF peptide sequencing with CAD fragmentation is efficient. However, in situations in which sufficient genome and protein sequence data are unavailable, mass spectrometric methods can be used for de novo protein sequencing. The mass measurement accuracy of MS provides a unique capability to... 

Mass spectral fragmentation patterns of alkyl and phenyl hydantoins have been investigated by means of labeling techniques (28—30), and similar studies have also been carried out for thiohydantoins (31,32). In all cases, breakdown of the hydantoin ring occurs by a-ftssion at C-4 with concomitant loss of carbon monoxide and an isocyanate molecule. In the case of aryl derivatives, the ease of formation of Ar—NCO is related to the electronic properties of the aryl ring substituents (33). Mass spectrometry has been used for identification of the phenylthiohydantoin derivatives formed from amino acids during peptide sequence determination by the Edman method (34). 

With the identities and amounts of amino acids known, the peptide is sequenced to find out in what order the amino acids are linked together. Much peptide sequencing is now done by mass spectrometry, using either electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) linked to a time-of-flight (TOF) mass analyzer, as described in Section 12.4. Also in common use is a chemical method of peptide sequencing called the Edman degradation. 

Tandem mass spectrometry (MS/MS) is a method for obtaining sequence and structural information by measurement of the mass-to-charge ratios of ionized molecules before and after dissociation reactions within a mass spectrometer which consists essentially of two mass spectrometers in tandem. In the first step, precursor ions are selected for further fragmentation by energy impact and interaction with a collision gas. The generated product ions can be analyzed by a second scan step. MS/MS measurements of peptides can be performed using electrospray or matrix-assisted laser desorption/ionization in combination with triple quadruple, ion trap, quadrupole-TOF (time-of-flight), TOF-TOF or ion cyclotron resonance MS. Tandem... 

Repeated addition of MDC to Q11 did occur, but the dominant product was Q11 with a single MDC. The fraction of Qll with higher numbers of attached MDC decreased for increasing MDC number. Separately, a lysine peptide that contained the bioactive RGD [73] sequence ( -dansyl-GLKGGRGDS-Am) was successfully TGase crosslinked with self-assembled Qll five distinct Qll-dansyl RGD were detected by mass spectrometry. 

Figure 5.11. Generic approaches to identify interacting proteins within complexes. The complex is isolated from cells by affinity purification using a tag sequence attached to a protein known to be in the complex. Alternatively, the complex can be immunprecipitated with an antibody to one of the proteins in the complex. The proteins are resolved by polyacrylamide gel electrophoresis, proteolyzed, and the mass of the resulting peptides is determined by mass spectrometry. Alternatively, the proteins can be proteolyzed and the resulting peptides resolved by liquid chromatography. The peptide masses are then determined by mass spectrometry and used for database searching to identify the component proteins.

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