For the characterization of protein sequences and post‐translational modifications by MS, the ‘top‐down’ proteomics approach utilizes molecular and fragment ion mass data obtained by ionizing and dissociating a protein in the mass spectrometer. This requires more complex instrumentation and methodology than the far more widely used ‘bottom‐up’ approach, which instead uses such data of peptides from the protein's digestion, but the top‐down data are far more specific. The ESI MS spectrum of a 14 protein mixture provides full separation of its molecular ions for MS/MS dissociation of the individual components. False‐positive rates for the identification of proteins are far lower with the top‐down approach, and quantitation of multiply modified isomers is more efficient. Bottom‐up proteolysis destroys the information on the size of the protein and the connectivities of the peptide fragments, but it has no size limit for protein digestion. In contrast, the top‐down approach has a ∼ 500 residue, ∼ 50 kDa limitation for the extensive molecular ion dissociation required. Basic studies indicate that this molecular ion intractability arises from greatly strengthened electrostatic interactions, such as hydrogen bonding, in the gas‐phase molecular ions. This limit is now greatly extended by variable thermal and collisional activation just after electrospray (‘prefolding dissociation’). This process can cleave 287 inter‐residue bonds in the termini of a 1314 residue (144 kDa) protein, specify previously unidentified disulfide bonds between eight of 27 cysteines in a 1714 residue (200 kDa) protein, and correct sequence predictions in two proteins, one of 2153 residues (229 kDa).
The most widely used modern mass spectrometers face severe performance limitations with molecules larger than a few kDa. For far larger biomolecules, a common practice has been to break these up chemically or enzymatically into fragments that are sufficiently small for the instrumentation available. With its many sophisticated recent enhancements, this "bottom-up" approach has proved highly valuable, such as for the rapid, routine identification and quantitation of DNA-predicted proteins in complex mixtures. Characterization of smaller molecules, however, has always measured the mass of the molecule and then that of its fragments. This "top-down" approach has been made possible for direct analysis of large biomolecules by the uniquely high (>10 5 ) mass resolving power and accuracy (~1 ppm) of the Fourier-transform mass spectrometer. For complex mixtures, isolation of a single component's molecular ions for MS/MS not only gives biomolecule identifications of far higher reliability, but directly characterizes sequence errors and posttranslational modifications. Protein sizes amenable for current MS/MS instrumentation are increased by a "middle-down" approach in which limited proteolysis forms large (e.g., 10 kDa) polypeptides that are then subjected to the top-down approach, or by "prefolding dissociation". The latter, which extends characterization to proteins >200 kDa, was made possible by greater understanding of how molecular ion tertiary structure evolves in the gas phase.
Building block of life? Infrared photodissociation spectroscopy shows at least six stable (Ser8+H)+ isomers. While retaining the chiral selectivity of the zwitterionic core, these vary mainly in H‐bonding of their peripheral OH groups (see picture). This could allow incorporation of any other amino acid to make these isomers unique for prebiotic selectivity of L‐amino acids for proteins.
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