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Protein sequencing by matrix-assisted laser desorption ionization-postsource decay-mass spectrometry analysis of the N-Tris(2,4,6-trimethoxyphenyl)phosphine-acetylated tryptic digests.
Anal Biochem. 1999 Mar 15; 268(2):305-17.AB

Abstract

We have recently reported a simple procedure by which low picomole quantities of peptides can be modified to the corresponding N-Tris(2, 4,6-trimethoxyphenyl)phosphonium-acetyl (TMPP-Ac) derivatives (Z. H Huang, J. Wu, D. A. Gage, and J. T. Watson, Anal. Chem. 69, 137-144, 1997). This modification significantly facilitates sequence interpretation by providing exclusively N-terminal product ions (mainly a-type ions) in the fast-atom bombardment-MS/MS and matrix-assisted laser desorption ionization-postsource decay(MALDI-PSD)-MS spectra. The TMPP-Ac derivatization approach has been extended now for the direct derivatization of tryptic digests originating from 1-5 microg of proteins with molecular weights from 10-120 kDa. Our new procedure involves tryptic digestion in aqueous solution buffered to pH 8-8.2 with phosphate or Tris-HCl, followed by reaction with TMPP-acetic acid N-hydroxysuccinimide ester (TMPP-AcOSu bromide, 2-4 nmol reagent/microg protein, rt, 20 min) to provide N-terminally derivatized products, while the epsilon-NH2 groups in lysine remain unchanged. The resultant derivatized peptide mixture or its partially separated HPLC fractions are subsequently analyzed by MALDI-PSD-MS using 0.5- to 1-pmol aliquots, giving rise to product ion spectra that are easily interpretable. As there is no need for material transfer and change of buffer media, the tandem enzymatic-chemical reaction/MS analysis process is usually carried out with very high throughput (digestion, 1 h; reaction, 1/3 h; HPLC, 1 h; MALDI-PSD, 3-4 fragments/h). This procedure will be of potential use for obtaining sequence information directly from mixtures or as an adjunct of peptide mass mapping to provide protein identification with high confidence.

Authors+Show Affiliations

Department of Chemistry, Michigan State University, East Lansing, Michigan, 48824, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

10075821

Citation

Huang, Z H., et al. "Protein Sequencing By Matrix-assisted Laser Desorption Ionization-postsource Decay-mass Spectrometry Analysis of the N-Tris(2,4,6-trimethoxyphenyl)phosphine-acetylated Tryptic Digests." Analytical Biochemistry, vol. 268, no. 2, 1999, pp. 305-17.
Huang ZH, Shen T, Wu J, et al. Protein sequencing by matrix-assisted laser desorption ionization-postsource decay-mass spectrometry analysis of the N-Tris(2,4,6-trimethoxyphenyl)phosphine-acetylated tryptic digests. Anal Biochem. 1999;268(2):305-17.
Huang, Z. H., Shen, T., Wu, J., Gage, D. A., & Watson, J. T. (1999). Protein sequencing by matrix-assisted laser desorption ionization-postsource decay-mass spectrometry analysis of the N-Tris(2,4,6-trimethoxyphenyl)phosphine-acetylated tryptic digests. Analytical Biochemistry, 268(2), 305-17.
Huang ZH, et al. Protein Sequencing By Matrix-assisted Laser Desorption Ionization-postsource Decay-mass Spectrometry Analysis of the N-Tris(2,4,6-trimethoxyphenyl)phosphine-acetylated Tryptic Digests. Anal Biochem. 1999 Mar 15;268(2):305-17. PubMed PMID: 10075821.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Protein sequencing by matrix-assisted laser desorption ionization-postsource decay-mass spectrometry analysis of the N-Tris(2,4,6-trimethoxyphenyl)phosphine-acetylated tryptic digests. AU - Huang,Z H, AU - Shen,T, AU - Wu,J, AU - Gage,D A, AU - Watson,J T, PY - 1999/3/17/pubmed PY - 1999/3/17/medline PY - 1999/3/17/entrez SP - 305 EP - 17 JF - Analytical biochemistry JO - Anal Biochem VL - 268 IS - 2 N2 - We have recently reported a simple procedure by which low picomole quantities of peptides can be modified to the corresponding N-Tris(2, 4,6-trimethoxyphenyl)phosphonium-acetyl (TMPP-Ac) derivatives (Z. H Huang, J. Wu, D. A. Gage, and J. T. Watson, Anal. Chem. 69, 137-144, 1997). This modification significantly facilitates sequence interpretation by providing exclusively N-terminal product ions (mainly a-type ions) in the fast-atom bombardment-MS/MS and matrix-assisted laser desorption ionization-postsource decay(MALDI-PSD)-MS spectra. The TMPP-Ac derivatization approach has been extended now for the direct derivatization of tryptic digests originating from 1-5 microg of proteins with molecular weights from 10-120 kDa. Our new procedure involves tryptic digestion in aqueous solution buffered to pH 8-8.2 with phosphate or Tris-HCl, followed by reaction with TMPP-acetic acid N-hydroxysuccinimide ester (TMPP-AcOSu bromide, 2-4 nmol reagent/microg protein, rt, 20 min) to provide N-terminally derivatized products, while the epsilon-NH2 groups in lysine remain unchanged. The resultant derivatized peptide mixture or its partially separated HPLC fractions are subsequently analyzed by MALDI-PSD-MS using 0.5- to 1-pmol aliquots, giving rise to product ion spectra that are easily interpretable. As there is no need for material transfer and change of buffer media, the tandem enzymatic-chemical reaction/MS analysis process is usually carried out with very high throughput (digestion, 1 h; reaction, 1/3 h; HPLC, 1 h; MALDI-PSD, 3-4 fragments/h). This procedure will be of potential use for obtaining sequence information directly from mixtures or as an adjunct of peptide mass mapping to provide protein identification with high confidence. SN - 0003-2697 UR - https://www.unboundmedicine.com/medline/citation/10075821/Protein_sequencing_by_matrix_assisted_laser_desorption_ionization_postsource_decay_mass_spectrometry_analysis_of_the_N_Tris_246_trimethoxyphenyl_phosphine_acetylated_tryptic_digests_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0003-2697(98)93085-8 DB - PRIME DP - Unbound Medicine ER -