Sequit: software forde novo peptide sequencing by matrix-assisted laser desorption/ionization post-source decay mass spectrometry

2004 ◽  
Vol 18 (8) ◽  
pp. 907-913 ◽  
Author(s):  
Rodion Demine ◽  
Peter Walden
Keyword(s):  
Mass Spectrometry ◽  
Laser Desorption ◽  
Peptide Sequencing ◽  
Laser Desorption Ionization ◽  
Post Source Decay ◽  
Desorption Ionization ◽  
Matrix Assisted Laser ◽  
Matrix Assisted Laser Desorption

Chemically-Assisted Fragmentation Combined with Multi-Dimensional Liquid Chromatography and Matrix-Assisted Laser Desorption/Ionization Post Source Decay, Matrix-Assisted Laser Desorption/Ionization Tandem Time-of-Flight or Matrix-Assisted Laser Desorption/Ionization Tandem Mass Spectrometry for Improved Sequencing of Tryptic Peptides

2005 ◽  
Vol 11 (2) ◽  
pp. 169-179 ◽  
Author(s):  
John Flensburg ◽  
Anders Tangen ◽  
Maria Prieto ◽  
Ulf Hellman ◽  
Henrik Wadensten
Keyword(s):  
Mass Spectrometry ◽  
De Novo ◽  
Laser Desorption ◽  
Peptide Sequencing ◽  
Laser Desorption Ionization ◽  
Post Source Decay ◽  
Tryptic Peptides ◽  
Desorption Ionization ◽  
Matrix Assisted Laser ◽  
Matrix Assisted Laser Desorption

Derivatization of tryptic peptides using an Ettan CAF matrix-assisted laser desorption/ionization (MALDI) sequencing kit in combination with MALDI-post source decay (PSD) is a fast, accurate and convenient way to obtain de novo or confirmative peptide sequencing data. CAF (chemically assisted fragmentation) is based on solid-phase derivatization using a new class of water stable sulfonation agents, which strongly improves PSD analysis and simplifies the interpretation of acquired spectra. The derivatization is performed on solid supports, ZipTipμC18, limiting the maximum peptide amount to 5 μg. By performing the derivatization in solution enabled the labeling of tryptic peptides derived from 100 μg of protein. To increase the number of peptides that could be sequenced, derivatized peptides were purified using multidimensional liquid chromatography (MDLC) prior to MALDI sequencing. Following the first dimension strong cation exchange (SCX) chromatography step, modified peptides were separated using reversed-phase chromatography (RPC). During the SCX clean up step, positively charged peptides are retained on the column while properly CAF-derivatized peptides (uncharged) are not. A moderately complex tryptic digest, prepared from six different proteins of equimolar amounts, was CAF-derivatized and purified by MDLC. Fractions from the second dimension nano RPC step were automatically sampled and on-line dispensed to MALDI sample plates and analyzed using MALDI mass spectrometry fragmentation techniques. All proteins in the derivatized protein mixture digest were readily identified using MALDI-PSD or MALDI tandem mass spectrometry (MS/MS). More than 40 peptides were unambiguously sequenced, representing a seven-fold increase in the number of sequenced peptides in comparison to when the CAF-derivatized protein mix digest was analyzed directly (no MDLC-separation) using MALDI-PSD. In conclusion, MDLC purification of CAF-derivatized peptides significantly increases the success rate for de novo and confirmative sequencing using various MALDI fragmentation techniques. This new approach is not only applicable to single protein digests but also to more complex digests and could, thus, be an alternative to electrospray ionization MS/MS for peptide sequencing.

Post-Source Decay Analysis of Dolastatin 10 and Dolastatin 15 by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

2002 ◽  
Vol 8 (4) ◽  
pp. 311-321 ◽  
Author(s):  
Nathalie Galéotti ◽  
Joël Poncet ◽  
Patrick Jouin
Keyword(s):  
Mass Spectrometry ◽  
Time Of Flight ◽  
Laser Desorption ◽  
Laser Desorption Ionization ◽  
Post Source Decay ◽  
Desorption Ionization ◽  
Flight Mass Spectrometry ◽  
Matrix Assisted Laser ◽  
Matrix Assisted Laser Desorption ◽  
Dolastatin 10

Dolastatin 10 and dolastatin 15 have been analyzed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry. Interestingly, in-source fragmentations could be easily detected for both compounds. Determination of the primary structure of both pseudopeptides could be done by using post-source decay (PSD) analysis. The molecular ion was used as the precursor ion in the case of dolastatin 10, whereas two in-source fragment ions and cationized (Na and K) precursors were selected in the case of dolastatin 15. An analogue of dolastatin 15 containing an amino alkyl linkage (compound 1) was also studied. Despite its close chemical structure its PSD spectrum exhibited a quite different pattern.

Changes in Post-Source Decay Fragmentation Behavior of Poly(Methyl Methacrylate) Polymers with Increasing Molecular Weight Studied by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

2003 ◽  
Vol 9 (3) ◽  
pp. 195-201 ◽  
Author(s):  
Olli Laine ◽  
Sarah Trimpin ◽  
Hans Joachim Räder ◽  
Klaus Müllen
Keyword(s):  
Mass Spectrometry ◽  
Molecular Weight ◽  
Laser Desorption ◽  
Laser Desorption Ionization ◽  
Post Source Decay ◽  
Ionization Time ◽  
Fragmentation Behavior ◽  
Desorption Ionization ◽  
Matrix Assisted Laser ◽  
Matrix Assisted Laser Desorption

In order to investigate the systematic changes in fragmentation behavior of poly(methyl methacrylate) (PMMA) with increasing molecular weight, alkali–metal cationized PMMA 20-mer, 60-mer and 100-mer were selected for post-source decay (PSD) fragmentation study by matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry. PMMA polymers were cationized with lithium, potassium and cesium cations to explore the influence of the cation size on the fragmentation behavior of the polymers. All PMMA polymers could be fragmented by MALDI-PSD and fragmentation of the MALDI ionized synthetic polymer of molecular weight 10 kDa is reported here for the first time. It was shown that an increasing molecular weight of the PMMA chain required an increase in the size of the cation to improve the intensity and the number of the fragments in the PSD spectrum. Some instrumental parameters had to be optimized prior to a successful PSD analysis of the largest PMMA polymers.

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