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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.
Eur J Mass Spectrom (Chichester). 2005; 11(2):169-79.EJ

Abstract

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, ZipTip(microC18, limiting the maximum peptide amount to 5 microg. By performing the derivatization in solution enabled the labeling of tryptic peptides derived from 100 microg 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.

Authors+Show Affiliations

Amersham Biosciences AB, GE Healthcare, Björkgatan 30, SE-751 84 Uppsala, Sweden. john.flensburg@ge.comNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

16046801

Citation

Flensburg, John, et al. "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." European Journal of Mass Spectrometry (Chichester, England), vol. 11, no. 2, 2005, pp. 169-79.
Flensburg J, Tangen A, Prieto M, et al. 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. Eur J Mass Spectrom (Chichester). 2005;11(2):169-79.
Flensburg, J., Tangen, A., Prieto, M., Hellman, U., & Wadensten, H. (2005). 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. European Journal of Mass Spectrometry (Chichester, England), 11(2), 169-79.
Flensburg J, et al. 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. Eur J Mass Spectrom (Chichester). 2005;11(2):169-79. PubMed PMID: 16046801.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Flensburg,John, AU - Tangen,Anders, AU - Prieto,Maria, AU - Hellman,Ulf, AU - Wadensten,Henrik, PY - 2005/7/28/pubmed PY - 2005/10/28/medline PY - 2005/7/28/entrez SP - 169 EP - 79 JF - European journal of mass spectrometry (Chichester, England) JO - Eur J Mass Spectrom (Chichester) VL - 11 IS - 2 N2 - 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, ZipTip(microC18, limiting the maximum peptide amount to 5 microg. By performing the derivatization in solution enabled the labeling of tryptic peptides derived from 100 microg 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. SN - 1469-0667 UR - https://www.unboundmedicine.com/medline/citation/16046801/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_ L2 - https://journals.sagepub.com/doi/10.1255/ejms.734?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub=pubmed DB - PRIME DP - Unbound Medicine ER -