Top-down protein sequencing

In the top-down method proteins are introduced into the mass spectrometer intact, i.e., without proteolysis. 

Individual amino acid residues are released from the end of the peptide chain using IRMPD or ECD. 

Ihe spectra generated are complex as the removal of each amino acid results in another set of multiply charged ions. Ihe number of atoms in the ions must be determined because of the 

FT-ICRMS is the most effective instrument because the resolution available facilitates spectrum interpretation by enabling charge state determination on highly charged ions. 

Get your % enzyme off my protein I have an FT-ICRMS and can do all the sequencing myself. 

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Macek, B., Waanders, L. F., Olsen, J. V., and Mann, M. (2006). Top-down protein sequencing and MS3 on a hybrid linear quadrupole ion trap-orbitrap mass spectrometer. Mol. Cell. Proteomics 5 949-958. 

Macek, B. Waanders, L.F. Olsen, J.V. Mann, M. Top-Down Protein Sequencing and MS on a Hybrid Linear Quad-... 

Multiply charged proteins can also be partially sequenced, and microsequences of proteins isolated from several microorganisms have been reported, accomplished with electrospray ionization and FTMS.23,90 Nonadjacent fragment ions may be used to identify bacterial proteins in these top-down strategies.91 In all cases these sequences could be related by bioinformatics to the parent species. An obvious extension would be to characterize proteins from intact microorganisms in this way. In at least one instance a microsequence has been obtained from a protein released from a contaminated intact bacteriophage sample (MS2) to provide a chemotaxonomic identification.77 This work was carried out in an ion trap mass spectrometer. 

Nemeth-Cawley, J.F., Tangarone, B.S., Rouse, J.C. (2003). Top Down characterization is a complementary technique to peptide sequencing for identifying protein species in complex mixtures. J. Proteome Res. 2, 495-505. 

When writing protein sequences, you write the amino terminus on the left. If you have to use the genetic code tables to figure out a protein sequence from the DNA sequence, it is not necessary to write down the complementary RNA sequence first it s the same as that of the sense strand (the one on top) with the Ts replaced by Us. 

Tandem mass spectrometry (see Chapter 3) can be applied to structural studies of various types of compounds, provided their molecular weights do not exceed approximately 2 to 4 kDa (there are also ways to analyze the sequence of larger molecules (proteins) by MS in the top-down strategy—see Figs. 6.2 and 6.3). In general, the larger... 

In recent years, a novel approach to protein identification emerged, called top-down sequencing. Here the entire nondigested protein is analyzed. Apart from accurate MW measurement, the protein ion is fragmented by the electron capture dissociation (ECD) method (see Chapter 3). This provides in-depth information on the sequence of protein. Such analysis can be performed only with FTICR instruments (see Section 2.2.6) that ensure high resolution and accuracy but, at the same time, they are exceptionally expensive. However, as very large ions are analyzed, even the high accuracy of FTICR is sometimes not sufficient, and it is recommended that such analyses are accompanied by more traditional bottom-up approaches. 

A growing number of researchers are focusing on the use of top-down proteomics, a relatively new approach compared to bottom-up, in which structure of proteins is studied through measurement of their intact mass followed by direct ion dissociation in the gas phase. The main advantages over the bottom-up approach are that higher sequence coverage is obtained, it permits... 

In comparison with the bottom-up approach, the top-down approach for de novo protein sequencing is faster and can be applied on proteins in mixture. However, for analysis of post-translational modifications, these two approaches are complementary and a combination of them is interesting. The bottom-up approach allows detection of low-stoichiometric modifications even if a high-stoichiometric modification is missed. Furthermore, enzymatic digestion leads to the loss of all connectivity information from the original protein species. On the other hand, the top-down approach allows observation of the global pattern of... 

The top-down approach, which sequences intact proteins by gas-phase fragmentation in the mass spectrometer, can also be used to characterize mutant proteins [103]. The position of the mutated amino acid can be unambiguously determined in each case by the characterization of product ions resulting from fragmentation on either side of the mutation site. 

The protein identification or sequence determination of a protein can be achieved using two different approaches top-down [22, 23] and bottom-up [24], A top-down experiment involves high-resolution measurement of an intact molecular weight and direct fragmentation of protein ions by tandem mass spectrometry (MS/MS) [25], This approach surveys an entire protein sequence with 100% coverage. Post-translational modifications such as glyco-... 

H. Zhai, X. Han, K. Breuker, F.W. McLafferty, Consecutive ion activation for top down MS Improved protein sequencing by nozzle-skimmer dissociation. Anal. Chem., 77 (2005) 5777. 

As such, the top-down strategy is a tool to recognize errors in the DNA-derived sequence, to identify (post-translational) modifications, or to evaluate modifications among complementary sets of the protein, e.g., in order to confirm the identity of a recombinant product. Further study with digestion and/or MS-MS then lows identification of the modification and its locatioa... 

The top-down strategy avoids a disadvantage of the bottom-up strategy in principle, it verifies the complete DNA-predicted sequence of the protein, as both the intact proteins and the sum of the fragments are measured. 

More recently, it has been demonstrated that the reagent anions used for either ETD or proton transfer can be derived from the same neutral compound. The radical anions used for ETD, [M]- , are converted into even-electron proton transfer reagent anions. [M + H]-, by changing the potential on the methane Cl source.1 9 This voltage switch can be acheived in milliseconds allowing for rapid sequential ion—ion reactions and opens up the possibility of top-down sequencing of intact proteins in RF ion traps. 

Top-down sequence analysis of the intact protein via ion-ion reactions 484... 

The molecular mass of an intact protein provides a frame within which the final structure must fit. The molecular mass information is an important analytical parameter required in many situations. Top-down proteomics relies on the molecular mass of intact proteins. This information is also needed to verify the correctness of the translated sequence of a protein and to identify point mutations, to find the extent of PTMs, and to determine the number of cysteine residues and disulfide bonds. ESI and MALDI are the methods of... 

A top-down sequence analysis approach has been developed in which sequence information on intact glycoproteins is obtained.121 First, the charge state of the ES-ionized protein is optimized through ion-ion reactions with [M - F and [M - CF3] anions. The CID spectrum of that precursor ion is well endowed with abundant sequence-specific ions, which are used to identify glycosylation sites. The top-down approach has the advantage that additional time- and sample-consuming purification and proteolytic cleavage experiments can be avoided. 

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