Plasma desorption peptides

R. A. Zubarev, V. D. Chivanov, P. Hakansson, B. U. R. Sundqvist, Peptide Sequencing by Partial Acid Hydrolysis and High Resolution Plasma Desorption Mass Spectrometry , Rapid Commun. Mass Spectrom. 1994, 8, 906-912. 

Plasma desorption <50,000 Simple to operate ability to analyze mixtures limited sequence information for pure, small peptides Very low resolution limits ability to detect structural variants not presently adapted for LC/MS long acquisition time, often >1 h... 

Further characterization of the remaining fraction in the low-sulfur keratin group in the ethanol-treated animals included analysis of the trypsin-released peptides by plasma desorption mass spectrometry. These results indicated the... 

Because C-terminal amides have often been reported in insect neuropeptides (8), a 33 residue C-terminal amide consistent with the sequence data was synthesized by solid-phase methods. The peptide was purified by HPLC and its structure confirmed by automated Edman degradation. Californium-252 time-of-flight plasma desorption mass spectrometry provided additional evidence for the structure via a very broad peak for the singly-charged molecular ion at m/z 3902-3906. Because the calculated MW of Hez-PBAN (3899.6, based on the most abundant ion in the isotope cluster) was seen to differ from that observed in the mass spectrum of the isolated native peptide by ca. 32, it was presumed that the native peptide had undergone oxidation of both its methionines to their respective sulfoxides during the course of its isolation and purification. 

Until 1988, the mass spectrometric analysis of peptides and proteins was difficult. Some results were achieved using (continuous-flow) fast-atom bombardment (FAB) and Cf plasma desorption. The major breakthrough in the characterization of proteins by mass spectrometry (MS) is due to the introduction of matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) in 1988. Currently, peptides and proteins form the compound class most intensively studied by MS. This is primarily due to the prominent role ESI-MS and MALDI-MS play in the field of proteomics. 

Chemical, electron field desorption, laser desorption, photon, plasma desorption, spark, and thermal ionization are all used as primary ionization processes. Secondary ionization is the term used to describe a process in which ions are ejected from a surface as a result of bombardment by a primary beam of atoms or ions. If low energy or soft ionization techniques are used, the mass of the target molecule can be determined. Advances in soft ionization techniques have extended the use of MS to the direct measurement of peptide and protein mass. Ionization at higher energy results in more extensive fragmentation of target molecules. 

Treatment of a peptide with 6M HC1 at 100-110 °C for 3-30 min and accurate mass measurement and immonium ion analysis (Section 4.7.1) using plasma desorption... 

New methods use combined HPLC/Mass spectrometry to identify modified amino acids. Purified recombinant human insulin-like growth factor separated into two peaks on reverse phase HPLC (Cl 8 column/acidified water) even though other methods indicated it was completely pure. Plasma desorption mass spectrometry of the individual peptides detected a single methionine sulfoxide molecule that was sufficient to decrease the hydrophobicity of the whole protein significantly. Most of the oxidation occurred when the secreted fusion protein was cleaved with hydroxylamine under not strictly anaerobic conditions, but about 5% occurred during die . coli fermentation. 

Early in the history of mass spectrometry (MS), large biomolecules were not analyzed because efficient methods to transport these molecules into the gas phase were unknown. Degradation typically occurred during vaporization of these nonvolatile molecules so that electron ionization of the intact molecular ion was not possible. Ionization by fast atom bombardment (FAB), field desorption (FD), secondary ionization mass spectrometry (SIMS), and plasma desorption (PD)" from the radioactive decay of Cf finally made the ionization and analysis of peptides possible. These latter techniques, although still used today, are not as popular as electrospray... 

In the past decade, mass spectrometry (MS) has become the method of choice for quality control of synthetic peptides. Historically, plasma desorption (PD) and fast atom bombardment (FAB) were the first ionization methods used for the mass analysis of nonderivatized peptides. More recently, electrospray ionization (ESI) MS and matrix-assisted laser desorption ionization (MALDI) MS have found widespread utility for peptide analysis. Both of the latter methods yield protonated molecules and, thus, provide direct molecular weight information. As will be covered later, ESI can be employed with a variety of mass analyzers, including quadrupole, magnetic sector, ion trap, and time-of-flight (TOF) analyzers. On the other hand,... 

Cleavage of the peptide from the resin and side-chain deprotection were carried out by treatment of the peptidyl resin with TFA/TlS/water (90 5 5) for 2.5 h. The crude peptide was analysed by HPLC (Figure 2d) and plasma desorption-mass spectrometry (PD-MS) [expected MH 1139.2, found 1139.1]. 

Plasma Desorption Mass Spectrometiy of Peptides and Proteins... 

Several mass spectrometric techniques including fast atom bombardment (FAB), plasma desorption (PD), matrix-assisted laser desorption/ionization (MALDI), and electrospray (ES) mass spectrometry (MS) are presently available for the analysis of peptides and proteins (Roepstorff and Richter, 1992). Of these techniques, mainly PDMS has gained footing in protein laboratories because the instrumentation is relatively cheap and simple to operate and because, taking advantage of a nitrocellulose matrix, it is compatible with most procedures in protein chemistry (Cotter, 1988 Roepstorff, 1989). Provided that the proper care is taken in the sample preparation procedure most peptides and small proteins (up to 10 kDa) are on a routine basis amenable to analysis by PDMS. Molecular mass information can be obtained with an accuracy of 0.1% or better. Structural information can be gained by application of successive biochemical or chemical procedures to the sample. 

Klarskov, K., Breddam, K., and Roepstorff, P. (1989) C-terminal sequence determination of peptides degraded with carboxypeptidases of different specificity and analyzed by Cf plasma desorption mass spectrometry. Anal. Biochem. 180, 28-37. 

Nielsen, P. F., Klarskov, K., Hojrup, P., and Roepstorff, P. (1988) Optimization of sample preparation for plasma desorption mass spectrometry of peptides and proteins. Biomed. Environm. Mass Spectrom. IS, 305-310. 

Roepstorff, P. (1989) Plasma desorption mass spectrometry of peptides and proteins. Acc. Chem. Res. 22, 421-427. 

The purified peptide has been used for additional studies of its physical properties. The peptide has certain unusual characteristics (such as its solvent solubility) that render it difficult to sequence. We found that the peptide has a free N-terminus. However, we have only been able to sequence through the first four amino acids (Phe-Leu-Cys-Ala...). After these residues the analysis stopped, probably due either to peptide substitutions or to the hydrophobic nature of this peptide. Current studies in our laboratory, therefore, are centered on using plasma desorption MS and microbore HPLC/dynamic FAB-MS to obtain physical data on this peptide. Such studies have shown that this peptide has a molecular mass of 3627.4 based on the singly charged molecular ion. 

Roepstorff P, Nielsen PF, Klarskov K, Hojrup P. Applications of plasma desorption mass spectrometry in peptide and protein chemistry. Biomed Environ Mass Spectrom. 1988 16 9-18. 

Much of the current interest in time-of-flight mass spectrometers is driven by instruments which desorb nonvolatile molecules (particularly peptides and other biological molecules) from surfaces. These methods include plasma desorption mass spectrometry (PDMS), laser desorption (LD), and matrix-assisted laser desorp-tion/ionization (MALDI), and they greatly simplify the design of time-of-flight mass spectrometers, since they effectively eliminate both the time- and spatial-distribution problems. 

While simple molecular weight measurements do not provide information on the amino acid sequences of peptides, they have been profoundly useful for verifying sequences which are inferred from the nucleic acid sequences of the genes encoding the peptide. In particular, they have been used to verify the sequences of peptides produced by recombinant techniques or by total chemical synthesis, or to reveal possible post-translational modifications. More specific information, however, can be obtained by comparative mass mapping of tryptic (or other enzymatic) digests. This approach is particularly useful when the molecular-ion mass exceeds the mass range of the plasma desorption technique. 

Ladder Sequencing. Because plasma desorption mass spectra commonly reveal only molecular ions, it has become common to obtain amino acid sequences using carboxy or amino-peptidases to generate a mixture of truncated peptides whose mass differences correspond to the amino acid residues. One example of this is shown in Figure 4.15, in which bradykinin has been digested directly on the nitrocellulose foil with carboxypeptidase... 

FIGURE 4.14 Plasma desorption mass spectra of the HPLC fractions containing peptide (a)aT6 and (b) an aT6 variant. (Reprinted with permission from reference 31). 

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