Extending Top-Down Mass Spectrometry to Proteins with Masses Greater Than 200 Kilodaltons

• DOI: 10.1126/science.1128868
• Publication Date: 2006-10-06T00:06:39Z
• Authors: Xuemei Han; Mi Jin; Kathrin Breuker; Fred W. McLafferty
• Keywords: Proteomics; 0301 basic medicine; Protein Folding; Spectrometry, Mass, Electrospray Ionization; Chemical Phenomena; Chemistry, Physical; Protein Conformation; Proteins; Complement C4; Mass Spectrometry; Peptide Fragments; Molecular Weight; 03 medical and health sciences; Humans; Amino Acid Sequence; Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor; Cysteine; Protein Processing, Post-Translational; Acyltransferases

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 ∼500-residue, ∼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).