Top-down identification and characterization of biomolecules by mass spectrometry

Abstract

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.

Stepwise evolution of protein native structure with electrospray into the gas phase, 10 −12 to 10 2 s

“…In this ''bottom-up'' approach, these protein fragment sequences are matched against the possible proteins predicted by their precursor DNA; matching peptide sequences can give up to 95% sequence coverage and 99% identification reliability. The ''top-down'' approach has far higher identification reliability and capabilities for locating sequence errors and posttranslational modifications (PTMs), although it requires instrumentation such as Fourier-transform MS of far higher resolving power and expense than instruments used previously for bottom-up proteomics (52)(53)(54). In this, individual molecular ions of a protein in a mixture are separated by MS-I and dissociated (MS-II) to give fragment ions indicative of that protein's sequence and PTM positions.…”

Differentiation of Glycan Diversity with Serial Affinity Column Set (SACS)

“…[1][2][3] Mass spectrometry (MS) is a significant methodology for proteomics in harmony with a wide variety of separation methods. Tandem mass spectrometry (MS/MS) is a major tool in protein identification.…”

“…Tandem mass spectrometry (MS/MS) is a major tool in protein identification. Mass spectrometers measure the mass to charge ratio of analytes; which include intact proteins and protein complexes, 3,4 fragment ions of protein ions, [5][6][7] peptides produced by enzymatically digested proteins, and fragment ions of selected peptide ions. [8][9][10] The application of mass spectrometry and MS/MS to proteomics takes advantage of the huge amount of genomic and proteomic data stored in databases.…”

Reproducible microwave-assisted acid hydrolysis of proteins using a household microwave oven and its combination with LC-ESI MS/MS for mapping protein sequences and modifications

“…In the top-down method, a protein ion is dissociated in a tandem mass spectrometer (MS/MS) and the fragment ions generated are interpreted to generate a stretch of amino acid sequence information. Sequence coverage by this method is dependent on the nature of the protein, ranging from a few residues to a full sequence [12]. In general, full sequence information is difficult to obtain for a protein with molecular mass of Ͼ20,000 Da [12].…”

“…Sequence coverage by this method is dependent on the nature of the protein, ranging from a few residues to a full sequence [12]. In general, full sequence information is difficult to obtain for a protein with molecular mass of Ͼ20,000 Da [12]. The bottom-up approach is a robust method for protein identification based on sequencing by MS/MS one or more peptides generated by chemical or enzymatic degradation of a protein.…”