Aspects of oligonucleotide and peptide sequencing with MALDI and electrospray mass spectrometry.
Biopolymer sequencing with mass spectrometry has become increasingly important and accessible with the development of matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). Here we examine the use of sequential digestion for the rapid identification of proteolytic fragments, in turn highlighting the general utility of enzymatic MALDI ladder sequencing and ESI tandem mass spectrometry. Analyses were performed on oligonucleotides ranging in size from 2 to 50 residues, on peptides ranging in size from 7 to 44 residues and on viral coat proteins. MALDI ladder sequencing using exonuclease digestion generated a uniform distribution of ions and provided complete sequence information on the oligonucleotides 2-30 nucleic acid residues long. Only partial sequence information was obtained on the longer oligonucleotides. C-terminal peptide ladder sequencing typically provided information from 4 to 7 amino acids into the peptide. Sequential digestion, or endoprotease followed by exoprotease exposure, was also successfully applied to a trypsin digest of viral proteins. Analysis of ladder sequenced peptides by LCMS generated less information than in the MALDI-MS analysis and ESI-MS2 normally provided partial sequence information on both the small oligonucleotides and peptides. In general, MALDI ladder sequencing offered information on a broader mass range of biopolymers than ESI-MS2 and was relatively straightforward to interpret, especially for oligonucleotides.
Owens
D R
DR
Department of Chemistry, Scripps Research Institute, La Jolla, CA 92037, USA.
Bothner
B
B
Phung
Q
Q
Harris
K
K
Siuzdak
G
G
D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000737:chemistry; D007304:Insect Viruses / Q000235:genetics; D009841:Oligonucleotides / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D017422:Sequence Analysis, DNA; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D017931:DNA Primers; D004279:DNA, Viral; D009841:Oligonucleotides; D010455:Peptides