Extending top-down mass spectrometry to proteins with masses greater than 200 kilodaltons.
For characterization of sequence and posttranslational modifications, molecular and fragment ion mass data from ionizing and dissociating a protein in the mass spectrometer are far more specific than are masses of peptides from the protein's digestion. We extend the approximately 500-residue, approximately 50-kilodalton (kD) dissociation limitation of this top-down methodology by using electrospray additives, heated vaporization, and separate noncovalent and covalent bond dissociation. This process can cleave 287 interresidue bonds in the termini of a 1314-residue (144-kD) protein, specify previously unidentified disulfide bonds between 8 of 27 cysteines in a 1714-residue (200-kD) protein, and correct sequence predictions in two proteins, one with 2153 residues (229 kD).
Han
Xuemei
X
Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, NY 14853, USA.
Jin
Mi
M
Breuker
Kathrin
K
McLafferty
Fred W
FW
D000217:Acyltransferases / Q000737:chemistry; D000595:Amino Acid Sequence; D019733:Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor / Q000737:chemistry; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003181:Complement C4 / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D003181:Complement C4; D010446:Peptide Fragments; D011506:Proteins; D000217:Acyltransferases; C044110:mycocerosic acid synthase; D019733:Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor; C022385:phosphoribosylformylglycinamidine synthetase; D003545:Cysteine