Peptides molecular weight determination

Covey, T.R. Bonner, R.F. Shushan, B.I. Henion, J.D. The Determination of Protein, Oligonucleotide, and Peptide Molecular Weights by lon-Spray-MS. Rapid Commun. Mass Spectrom. 1988, 2, 249-256. 

Different authors used RP-HPLC and UV detection to monitor peptide formation during cheese ripening [174-178], providing valuable information about proteolysis. When large hydrophobic peptide need to be separated an lEC represents the best choice [179]. Nevertheless, the identification of these peptides is essential for the complete understanding of the proteolytic process. The peptides eluted from the LC column can be subjected to ESl-MS for molecular weight determination and MS/MS for amino acid sequence determination, which allow rapid peptide identification [172]. HPLC-ESl-MS and MS/MS techniques have been successfully used for peptide mass fingerprint purposes for sequence analysis of purified albumin from Theobroma cacao seeds [180,181]. 

Fast atom bombardment mass spectrometry (FABMS) has become an important addition to the ionization techniques available to the analytical chemist in recent years. It has been particularly useful in a number of diverse applications which include molecular weight determinations at high mass, peptide and oligosaccharide sequencing, structural analysis of organic compounds, determination of salts and metal complexes, and the analysis of ionic species in aqueous solutions. This paper will focus on some aspects of the quantitative measurement of ionic species in solution. The reader is referred to a more comprehensive review for more details of some of the examples given here as well as other applications (1). 

The advent of FAB mass spectrometry has allowed the routine molecular weight determination of polar molecules, without derivatization, up to ca 3,000 Daltons, and in exceptional cases, within 1 mass unit to the region of 8,000 Daltons. This advance, coupled with FAB fragmentation, and enzymic digestion techniques, has allowed the rapid solution of a number of problems in protein and peptide chemistry - problems which were hitherto rather difficult to solve. Examples are given. 

A sample size of approximately 0.1 nanomoles is generally sufficient for molecular weight determination in either the positive or negative ion mode, but does not normally allow the sequence of amino acids to be determined. Larger sample sizes, typically between 1 and 5 nanomoles, afford some sequence information. Sequence ions are observed in the positive and negative ion modes from both N- and C-termini of the peptide and this may enable the complete sequence of the peptide to be determined. 

A molecular weight determination has shown that an unknown peptide is a pentapeptide, and an amino acid analysis shows that it contains the following residues one Gly, two Ala, one Met, one Phe. Treatment of the original pentapeptide with carboxypeptidase gives alanine as the first free amino acid released. Sequential treatment of the pentapeptide with phenyl isothiocyanate followed by mild hydrolysis gives the following derivatives ... 

T.R. Covey, R.F. Bonner, B.I. Shushan, J.D. Henion, The determination of protein, oligonucleotide and peptide molecular weights by ESI-MS, Rapid Commun. Mass Spectrom., 2 (1988) 249. 

Capillary isoelectric focusing coupled to mass spectrometry has gained popularity in recent years (by analogy to two-dimensional gel electrophoresis). Additional information obtained from mass spectrometry includes not only precise molecular-weight determination but also the possiblity for peptide sequencing. Analysis of hemoglobin variants, recombinant proteins, and monoclonal antibodies have been demonstrated [1,7,8]. 

Mass spectrometry can determine the molecular weights of peptides and proteins with mass accuracies orders of magnitude better than the molecular weights determined by gel electrophoresis. It is important to note that in determining molecular... 

Figure 3.3 Separation and molecular weight determination of peptides by mass spectrometry. Intensity indicates the number of molecules of a peptide, whereas the time of flight indicates the molecular weight of a peptide.

Bacitracin B has been purified by countercurrent distribution, but it seems that this substance also is slowly transformed. Molecular weight determination and hydrolysis have given a value for a peptide containing 13 amino acids, consisting of all the amino acids of bacitracin A and an additional valine. Although some partial sequences are known, the final structure has yet to be determined. Whether the other members of the bacitracin group are real entities or transformation products remains to be seen. 

Actually deciphering the complete sequence from the MS fragmentation pattern is an extremely complex task that is beyond the scope of this chapter. However, verification of a proposed sequence should be possible, even for facilities just being introduced to peptide analysis by MS. In the case of the ABRF study described above, with the availability of MS/MS data it should have been obvious to the participating laboratories that the requested peptide had not been synthesized. With the results of this study as an example, it can be concluded that when other quality assurance mechanisms are in place in a peptide synthesis laboratory, molecular weight determination by either MALDI-TOF or ESI-MS can provide an excellent means of verifying the integrity of a synthetic product. When questions do arise about a specific peptide, MALDI-PSD and ESI-MS/MS then can permit rapid determination of sequence information that should yield valuable clues about the nature of a synthetic sequence. 

The ehmination of peptides and proteins can occur unspecifically nearly everywhere in the body or can be limited to a specific organ or tissue. Locations of intensive peptide and protein metabolism are liver, kidneys, gastrointestinal tissue, and also blood and other body tissues. Molecular weight determines the major metabolism site as well as the predominant degradation process [13, 42] (Table 1). 

Another appUcation is in the recovery of peptides after chemical modification, e.g. to remove the alkylating agent after alkylation (Fig. 11.2.5). Accurate molecular weight determination, even with a narrow range of standards, is not reliable unless carried out in the presence of strong denaturants. 

The spectrum in Fig. 4.9 demonstrates that MALDI-TOF is a powerful method for accurate molecular weight determination of peptides and proteins. As there is almost no fragmentation, mixtures of peptides and proteins can be analysed without having to separate the compounds prior to analysis. In this respect, MALDI-TOF has to be regarded as a very fast separation method and is in many ways more powerful than chromatography or electrophoresis. In Fig. 4.10, a spectrum of low fat bovine milk is shown. The milk sample was added to the matrix without any pre-treatment and the different components present in the sample are resolved in the obtained mass spectrum. 

ESI is suitable for almost all kinds of biomolecules, as long as they are polar and soluble in a solvent system that can be used for spraying. Peptides, proteins, carbohydrates, DNA fragments and lipids are all commonly analysed via ESI-MS. Molecular weight determination is one of the main applications. Eurthermore, sequencing of peptides and DNA fragments (section 6.3) is possible with ESI connected to a tandem mass spectrometer (ESI-MS/MS). 

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