Liquid chromatographic–high-resolution mass spectrometric and tandem mass spectrometric identification of synthetic peptides using electrospray ionization

D’Agostino, Paul A.; Hancock, James R.; Provost, Lionel R.; Semchuk, Paul D.; Hodges, R. S.

doi:10.1016/s0021-9673(97)01104-7

Cited by 17 publications

(11 citation statements)

References 27 publications

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“…[33][34][35] Traditionally, magnetic sector mass spectrometers have been applied for accurate mass measurements of low partsper-million (ppm) accuracy. Recently, ESI with magnetic sector instruments has been used for even small molecule (less than 1000 Da) accurate mass analysis, [36][37][38] where ionization methods such as electron ionization (EI), chemical ionization (CI), and fast atom bombardment (FAB) have been traditionally used. The ppm accuracy measurements can be extended to biomolecules such as large peptides and small proteins.…”

Section: Discussionmentioning

confidence: 99%

“…The ppm accuracy measurements can be extended to biomolecules such as large peptides and small proteins. 12,39 The applicability of magnetic sector mass spectrometry to biomolecular research has been further augmented by capabilities for on-line separation methodologies, such as high performance liquid chromatography 37,40 and capillary electrophoresis. 41,42 With high efficiency CAD analyzers, such as orthogonal TOF and ion trap stages for sensitive structural elucidation studies, such hybrid systems have become extremely versatile and powerful mass spectrometers.…”

Section: Discussionmentioning

confidence: 99%

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Magnetic sector-ion trap mass spectrometry with electrospray ionization for high sensitivity peptide sequencing

Loo¹,

Muenster²

1999

Rapid Commun. Mass Spectrom.

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A hybrid mass spectrometer composed of a high resolution double focusing instrument (electrostatic analyzer-magnetic sector, EB) and an ion trap analyzer (T) exhibits high sensitivity performance for peptide sequencing with electrospray ionization (ESI). MS 2 and MS 3 experiments for multiply charged tryptic peptides and larger peptides (e.g., melittin, 2.8 kDa) generate sequence-informative product ions. Collisionally activated dissociation (CAD) of selected precursor ions can also be performed in the interface between the double focusing analyzer and the ion trap (transfer octapole region) to generate product ions. With a low-flow micro-ESI source, which can deliver analyte solution to the source at a flowrate of 10-200 nL/min, tandem mass spectra can be obtained from sub-fmol amounts of melittin. The high resolving power of the MS-I stage combined with the efficiency of the ion trap stage allows for high resolution precursor ion selection with subsequent highly sensitive tandem mass spectrometry (MS/MS) analysis. # 1999 John Wiley & Sons, Ltd. Received 2 October 1998; Revised 4 November 1998; Accepted 5 November 1998 Modern mass spectrometry provides a highly sensitive and rapid method for sequencing of polypeptides, especially with the development of ionization methods such as electrospray ionization (ESI).1 Mass analyzers have also undergone a dramatic transformation in recent years, as improvements in high performance instruments such as Fourier transform ion cyclotron resonance systems (FTICRMS) 2 and ion trap mass spectrometers [3][4][5] and new geometries such as the quadrupole-time-of-flight (Q-TOF) 6,7 have been developed. These enhancements in tandem mass spectrometry (MS/MS) sensitivity have mirrored the increasing demand for protein sequencing for biomedical research projects. The identification of proteins expressed by organisms, cell, and tissues, in different environmental conditions (proteome analysis), often relies on the sequence determination provided by MS/MS. 8-10Mass spectrometers based on magnetic (B) and electrostatic (E) analyzers operating at 5-10 kV have been interfaced with ESI sources.11-14 High resolving power and high m/z range (typically to 10 000) are features and advantages of such magnetic sector systems over the more common analyzers (e.g., ion traps and quadrupoles) fitted with ESI. For MS/MS analysis, methods relying on linked scans at a constant B/E ratio can be used for peptide sequencing with ESI, 15 but suffer from poor sensitivity and poor parent ion resolution. Instruments based on two double focusing systems have high precursor and product ion resolution, but require rather large footprints and may be cost-prohibitive for most laboratories. Other analyzers have been interfaced to magnetic sector instruments for MS/MS experiments. These hybrid systems have been well described by Yost and Boyd 16 and others. 17 Tandem quadrupoles (consisting of a radiofrequency-only collision quadrupole cell and a quadrupole mass analyzer, qQ) have been interfaced to sector-based ...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Magnetic sector-ion trap mass spectrometry with electrospray ionization for high sensitivity peptide sequencing

Loo¹,

Muenster²

1999

Rapid Commun. Mass Spectrom.

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“…Liquid chromatography coupled with electrospray mass spectrometry has proved to be highly efficient for fast and reliable analysis and characterization of peptides in complex crude mixtures [27,28,29,30,31,32,33,34,35,36].…”

Section: Introductionmentioning

confidence: 99%

Liquid chromatography?mass spectrometry approach for the characterization and purification of crude synthetic peptide hormones

Sanz‐Nebot

Benavente

Toro

et al. 2003

Analytical and Bioanalytical Chemistry

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In this study, conditions for the optimal separation by LC-UV and characterisation by LC-ES-MS of crude mixtures generated during SPPS of several peptide hormones are compiled. The linear solvation energy relationship (LSER) methodology has been used to predict the retention of the target peptides and their side products and then to develop a separation LC methodology with applicability on both the analytical and preparative scale. Identification of these side products by LC-ES-MS analysis has been made on the basis of their calculated molecular masses. This method may be regarded as a key tool for the optimisation of the synthetic procedures and for complying with regulatory agencies' requirements before commercialisation of a safe and effective peptide-based pharmaceutical drug.

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“…Therefore, everything that can be learned about the binding and catalysis of the active site of this new ZIP enzyme will aid in the design of site-directed inhibitors. 16,17,18,19,20 . In this paper, we describe a method to separate and characterise fragments of the reactions between this new ZIP enzyme and its substrates using LC-MS. We report on where cleavage takes place in the peptides and on the cleavage products formed.…”

Section: Introductionmentioning

confidence: 99%

Characterisation of the active site of a newly-discovered and potentially significant post-proline cleaving endopeptidase called ZIP using LC-UV-MS

McMahon

Collins

O’Connor

2003

Analyst

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Abbreviations AbstractThere are enzymes that specifically recognise the amino acid proline within peptides and proteins that are called post-proline cleaving enzymes. Many of them are implicated in neurodegenerative disorders and psychiatric diseases. ZIP is a newly-discovered one of these peptidases. In this work, it has been purified from bovine serum and subjected to various analytical studies in order to characterise it. A series of reactions between synthesised peptides and ZIP were carried out in order to elucidate the size and specificity of the active site of the enzyme. On-line LC-MS was carried out on samples before and * corresponding author Dr. Gillian McMahon:Tel.: -353-1-7005914, fax: -353-1-7008021, e-mail: gillian.mcmahon@dcu.ie after incubation and the results obtained allowed us to detect if cleavage of the peptides was taking place, and if so, where in the peptide chain. It was found that the enzyme has a preference for another larger, bulky amino acid to follow the proline and that little or no cleavage was observed when polar acidic or other small amino acids were in that location. In terms of the size of the active site, the endopeptidase was found to have optimum activity when there were two more amino acids after the proline, with a fall-off in activity detected for the longer peptides. Data from kinetic studies confirmed the LC-MS results. The methodology described in this paper, which is a combination of LC separation and UV/MS detection, is required for the accurate monitoring of the reactions between the peptidase and its peptide substrates and for analysis of the products of such enzyme-peptide reactions. This work will assist in the design of site-directed inhibitors for new drug therapies.

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Liquid chromatographic–high-resolution mass spectrometric and tandem mass spectrometric identification of synthetic peptides using electrospray ionization

Cited by 17 publications

References 27 publications

Magnetic sector-ion trap mass spectrometry with electrospray ionization for high sensitivity peptide sequencing

Magnetic sector-ion trap mass spectrometry with electrospray ionization for high sensitivity peptide sequencing

Liquid chromatography?mass spectrometry approach for the characterization and purification of crude synthetic peptide hormones

Characterisation of the active site of a newly-discovered and potentially significant post-proline cleaving endopeptidase called ZIP using LC-UV-MS

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