Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of 4-sulfophenyl isothiocyanate-derivatized peptides on AnchorChip sample supports using the sodium-tolerant matrix 2,4,6-trihydroxyacetophenone and diammonium citrate.
The reagent 4-sulfophenyl isothiocyanate (SPITC) is an effective, stable, and inexpensive alternative to commercially available reagents used in the N-terminal sulfonation of peptides for enhanced postsource decay (PSD) in matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analyses. However, suppression of ionization of sulfonated peptides due to sample and matrix contaminants such as sodium can be a problem when using prestructured MALDI target sample supports, such as the Bruker Daltonics AnchorChip. We show that use of the salt-tolerant matrix 2,4,6-trihydroxyacetophenone containing diammonium citrate (THAP/DAC) as an alternative to alpha-cyanohydroxycinnamic acid (HCCA) reduces the need for extensive washing of ZipTip-bound peptides or additional on-target sample clean-up steps. Use of the THAP/DAC matrix results in selective ionization of sulfonated peptides with greater peptide coverage, as well as detection of higher mass derivatized peptides, than was observed for HCCA or THAP alone. The THAP/DAC matrix is quite tolerant of sodium contamination, with SPITC-peptides detectable in preparations containing up to 50 mM NaCl. In addition, THAP/DAC matrix was found to promote efficient PSD fragmentation of sulfonated peptides. We demonstrated the utility of using the THAP/DAC MALDI matrix for peptide sequencing with DNA polymerase beta tryptic peptide mixture, as well as tryptic peptides derived from Xiphophorus maculatus brain extract proteins previously separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE).
Oehlers
Leon P
LP
Molecular Biosciences Research Group, Department of Chemistry and Biochemistry, Texas State University, San Marcos, TX 78666, USA.
Perez
Amy N
AN
Walter
Ronald B
RB
D000098:Acetophenones / Q000032:analysis / Q000737:chemistry*; D000818:Animals; D001921:Brain / Q000378:metabolism*; D003532:Cyprinodontiformes; D019951:DNA Polymerase beta / Q000032:analysis / Q000737:chemistry*; D004361:Drug Tolerance; D017879:Isothiocyanates / Q000032:analysis / Q000737:chemistry*; D008571:Men; D009479:Neuropeptides / Q000032:analysis / Q000737:chemistry*; D040081:Protein Array Analysis / Q000379:methods; D020539:Sequence Analysis, Protein / Q000379:methods*; D012964:Sodium / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000098:Acetophenones; D017879:Isothiocyanates; D009479:Neuropeptides; C107067:2,4,6-trihydroxyacetophenone; D012964:Sodium; D019951:DNA Polymerase beta