Mass spectrometry synthetic peptides

Arttamangkul, S., Arbogast, B., Barofsky, D. and Aldrich, J.V. (1997) Characterization of synthetic peptide byproducts from cyclization reactions using on-line HPLC-ion spray and tandem mass spectrometry. Lett. Peptide Sci., 3, 357-70. 

S. Beranova-Giorgianni and D. M. Desiderio. Fast Atom Bombardment Mass Spectrometry of Synthetic Peptides. In Methods in Enzymology Solid-Phase Peptide Synthesis, ed. G. B. Fields. Methods in Enzymology 289. Academic Press, San Diego, 1997, 478-499. 

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. 

When air oxidation of the reduced p-conotoxin GIIIB (18) was carried out in 0.1 M NHtOAc buffer (pH 7.5) at 0.01 mM peptide concentration and at 10 °C, three major products, isomers 15,16, and 17 were produced after 40 hours in a ratio of 1 4 3 (Figure 2). 86 The disulfide structures of each isomer were determined by enzymatic digestion followed by amino acid analyses, mass spectrometry, sequence analyses, as well as by the synthetic approach (Scheme 10). 

Mass spectrometry has gained widespread application for the characterization of synthetic peptides and has led, in combination with HPLC, to the identification of accompanying byproducts such as polysulfides 17 ... 

Presently, FAB-MS spectra are routinely used to characterize synthetic tyrosine O-sulfate peptides.152,57,63-671 Since partial hydrolysis of the sulfate ester occurs in the gas phase, quantification of the tyrosine O-sulfate residue by mass spectrometry is not possible, but combined with one-peak assignment in HPLC, FAB-MS represents a powerful analytical tool. On the other hand, partial hydrolysis in the gas phase excludes the presence of sul-fonated species which should be perfectly stable. In early studies the presence of such species were excluded by quantitative recovery of tyrosine upon acid hydrolysis or upon hydrolysis with arylsulfatase.1361 Recently, even MALDI-TOF-MS spectra of CCK-peptides1441 and of conotoxins a-PnIA and a-PnlB 138 were reported which show that in the positive-ion mode the [M + H-S03]+ ions represent the base peaks, while in the negative-ion mode, [M-H]-ions consistently correspond to the base peaks. In the CCK peptides intramolecular salt bridging of the sulfate hemi-ester with proximal positive charges of arginine or lysine side chains was found to reduce the extent of hydrolysis in the gas phase significantly.144,1491... 

Great care has to be taken in the analytical characterization of synthetic cyclic peptides.[73] The major side reactions during cyclization are epimerization of the C-terminal amino acid residue and cyclodimerization. Cyclodimers can be detected by mass spectrometry, although the analysis is not trivial, because artifacts do occur in some ionization techniques such as ES-MS as a result of aggregation.1 1 Ll 121 Real dimers can be detected as double-charged particles with mlz values identical to the cyclic monomers, but with a mass difference of 0.5 amu in the resolved isotope signals. The mass difference of the corresponding monomer is 1 amu. The cyclodimerization has received some attention as a direct method for the synthesis of C2-symmetrical cyclic peptides.[62 67 94113 115]... 

Beranova-Giorgianni, S., Desiderio, D.M. (1997). Fast atom bombardment mass spectrometry of synthetic peptides. Methods Enzymol., 289, 478 499. 

Laser desorption Fourier transform mass spectrometry (LD-FTMS) results from a series of peptides and polymers are presented. Successful production of molecular ions of peptides with masses up to 2000 amu is demonstrated. The amount of structurally useful fragmentation diminishes rapidly with increasing mass. Preliminary results of laser photodissociation experiments in an attempt to increase the available structural information are also presented. The synthetic biopolymer poly(phenylalanine) is used as a model for higher molecular weight peptides and produces ions approaching m/z 4000. Current instrument resolution limits are demonstrated utilizing a polyethylene-glycol) polymer, with unit mass resolution obtainable to almost 4000 amu. 

Smart SS, Mason TJ, Bennell PS, Maeji NJ, Geysen HM, High-throughput purity estimation and characterization of synthetic peptides by electrospray mass spectrometry, Int. J. Peptide. Protein. Res., 47 47-55, 1996. 

The peptide concentration in the individual specimens is determined by radioimmunoassay, using a synthetic reference peptide for the standard curve. Several commercial assays are now available for hypothalamic peptides. An alternative method is the determination of peptide content in tissue extracts by an HPLC method or by mass spectrometry. 

Because anurans, including L. splendida, often breed in water, the peptide components of the secretions of the paratoid and rostral glands were purified directly by HPLC. The active component was sequenced with the aid of electrospray ionization mass spectrometry (ESI-MS) and given the name splendipherin (17).76 The structure of this 25-amino acid peptide subsequently was confirmed by comparison with a synthetically prepared sample, which further established that all amino acids were of the L-configuration. 

Mass spectrometry also can play an important role in the verification of the structure and purity of synthetic peptides [104-106], The development of automatic synthesizers has made the production of synthetic peptides increasingly easier. A large number of errors, however, may occur during or after the synthesis (see Table 8.7) the majority of which are... 

Following the first studies of J.J. Thomson (1912), mass spectrometry has undergone countless improvements. Since 1958, gas chromatography coupled with mass spectrometry has revolutionized the analysis of volatile compounds. Another revolution occurred in the 1980s when the technique became available for the study of non-volatile compounds such as peptides, oligosaccharides, phospholipids, bile salts, etc. From the discoveries of electrospray and matrix-assisted laser desorption in the late 1980s, compounds with molecular masses exceeding several hundred thousands of daltons, such as synthetic polymers, proteins, gly-cans and polynucleotides, have been analysed by mass spectrometry. 

Similar mass spectrometry experiments with pure human and mouse transferrin (Li et al., 2008c), and with human kinesin showed that the OP label was consistently on tyrosine (Table 56.1). Studies with human plasma identified OP labeling on tyrosine in apolipoprotein and alpha-2-glyco-protein. Aggressive treatment of hiunan albumin with FP-biotin and chlorpyrifos oxon led to identification of seven OP-labeled tyrosines (Ding et al, 2008). Finally, we found that synthetic peptides made a covalent bond with DFP, chlorpyrifos oxon, and dichlorvos (Table 56.1). Mass spectrometry conclusively proved that the OP was attached to tyrosine. 

Protein Sequencing, Peptide Synthesis, Amino Add Analysis and Mass Spectrometry - Methods for protein modification, proteolysis, RP-HPLC peptide purification and automated Edman degradation are well documented (7,10,14) as are methods for FMOC synthesis and myristylation of synthetic peptides (15) and amino acid analysis (16). 

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