Top Down versus Bottom Up Protein Characterization by Tandem High-Resolution Mass Spectrometry

Abstract

Characterization of larger proteins by mass spectrometry (MS) is especially promising because the information complements that of classical techniques and can be obtained on as little as 10-17 mol of protein. Using MS to localize errors in the DNA-derived sequence or modifications (posttranslational, derivatized active sites, etc.) usually involves extensive proteolysis to yield peptides of <3 kDa, with separation and MS/MS to compare their sequences to those expected (the “bottom up” approach). In contrast, an alternative “top down” approach limits the dissociation (proteolysis or MS/MS) to yield larger products from which a small set of complementary peptides can be found whose masses sum to those of the molecule. Thus a disagreement with the predicted molecular mass can be localized to a fragment(s) without examining all others, with further dissociation of the fragments in the same way providing further localization. Using carbonic anhydrase (29 kDa) as an example, Fourier transform mass spectrometry is unusually effective for the bottom up approach, in that a single spectrum of an extensive chymotryptic digest identifies 64 expected peptides, but these only cover 95% of the sequence; 20 fragment masses are unassigned so that any set whose masses sum to that of the molecule would be misleading. Extensive Lys-C dissociation yields 17 peptides, 23 unassigned masses, and 96% coverage. In the contrasting “top down” approach, less extensive initial dissociation by Lys-C, MS/MS, or CNBr in each case provides 100% coverage, so that modified protein fragment(s) could easily be recognized among the complementary sets. MS/MS of such a fragment or more extensive proteolysis provide further localization of the modification. The combined methods cleaved 137 of the 258 amide bonds between residues.

Top down characterization of secreted proteins from Mycobacterium tuberculosis by electron capture dissociation mass spectrometry

“…After being frozen in Ϫ80°C and thawed, any precipitate was resuspended in 6 M urea. (Foster City, CA) ESI/MS and MS/MS data were acquired on a 6 T modified FTMS with nano-electrospray [22][23][24][25][26]. Specific ions were isolated using stored waveform inverse Fourier-transform [32], followed by sustained off-resonance irradiation collisionally-activated dissociation (CAD) [33,34], infrared multiphoton dissociation (IRMPD) [35], and activated ion ECD [26].…”

“…Although rarely observed in eubacteria, in immunology the glycosylated proteins may contribute to the interaction with the mammalian cells during infection [20,21]. This report utilizes the top-down MS/MS approach [22,23] to identify proteins secreted into the extracellular milieu, in which mixed protein components are ionized and separated directly by Fourier transform MS and then dissociated MS/MS to provide fragment masses for matching against the DNA predicted sequence and for structural characterization of posttranslational modifications. This study also illustrates the applicability of the recently developed MS/MS technique of electron capture dissociation (ECD) [24 -26].…”

A preferred AMPK phosphorylation site adjacent to the inhibitory loop of cardiac and skeletal troponin I

“…The top-down MS strategy allows analysis of intact proteins to obtain a comprehensive assessment and localization of post-translational modifications (PTMs) and point mutations with full sequence coverage and without a priori knowledge. [27][28][29][30][31][32][33][34][35][36][37] It starts with measuring molecular weights of intact proteins followed by fragmentation of the protein in the mass spectrometer with tandem mass spectrometry (MS/MS) such as electron capture dissociation (ECD) 38 to obtain sequence information. ECD has been demonstrated to be especially valuable for mapping phosphorylation sites, because it preserves the labile PTMs during MS/MS process.…”

Characterization of a new qQq-FTICR mass spectrometer for post-translational modification analysis and top-down tandem mass spectrometry of whole proteins

“…Another promising application of FTICR MS is the ability to perform top-down protein analysis, i.e., the fragmentation°of°intact°proteins° [51].°The°ability°to°start MS n analysis with the intact protein, by definition, yields 100% sequence coverage, enabling the complete characterization of the protein and any associated post-translational modifications. This is of paramount importance when carrying out phosphopeptide mapping studies.…”