Proteins top-down

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. 

The combination of this top-down proteomics approach, which generates information on the structure of the intact protein, with a bottom-up approach for protein identification (using MS/MS data of tryptic peptides from the collected fractions) has been particularly useful for identifying posttranslational modifications, cotransla-tional processing, and proteolytic modifications in a number of proteins. Examples from our work will be shown to illustrate this hybrid methodology for proteomics analysis. 

The ability to resolve and characterize complicated protein mixtures by the combination of 2DLC and online mass spectrometry permits the combination of sample fractionation/simplification, top-down protein mass information, and bottom-up peptide level studies. In our lab, the simplified fractions generated by 2D(IEX-RP)LC are digested and analyzed using common peptide-level analysis approaches, including peptide mass fingerprinting (Henzel et al., 1993 Mann et al., 1993), matrix-assisted laser desorption/ionization (MALDI) QTOF MS/MS (Millea et al., 2006), and various capillary LC/MS/MS methodologies (e.g., Ducret et al., 1998). 

The ongoing development of top-down protein MS/MS capabilities (MS/MS) should prove quite valuable to researchers looking to identify and characterize proteins fractionated by 2DLC separations. Such methods are currently limited by restrictions on the maximum size of proteins analyzed, as well as analysis time-requirements that limit coupling of these methods with online LC analysis. Investigators from labs, such as Kelleher, McLafferty, Hunt (Coon et al., 2005 Meng et al., 2005 Han et al., 2006), and others are rapidly addressing these issues, and their methods will likely be adopted by many other researchers over the next few years. 

Han, X., Jin, M., Breuker, K., McLafferty, F.W. (2006). Extending top-down mass spectrometry to proteins with masses greater than 200 kilodaltons. Science 314, 109-112. 

LeDuc, R.D., Taylor, G.K., Kim, Y.B., Januszyk, T.E., Bynum, L.H., Sola, J.V., Garavelli, J.S., Kelleher, N.L. (2004). ProSight PTM an integrated environment for protein identification and characterization by top-down mass spectrometry. Nucleic Acids Res. 32, W340-W345. 

Millea, K.M., Krull, I.S., Cohen, S.A., Gebler, J.C., Berger, S.J. (2006). Integration of multidimensional chromatographic protein separations with a combined top-down and bottom-up proteomic strategy. J. Proteome. Res. 5, 135-146. 

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. 

X. Han, M. Jin, K. Breuker, and F. W. McLafferty. Extending Top-Down Mass Spectrometry to Proteins with Masses Greater Than 200 Kilodaltons. Science, 314(2006) 109-112. 

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. 

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. 

Figure 4.5 NCE/ESI QTOF-MS and tandem MS electropherograms of DNA-binding domain of human TTAGGG repeat binding factor 2 (hTRF2 DBD) protein. (A) Mass electropherogram of hTRF2 DBD [M+ 5H]S+ ion at m/z 1509, and (B) top-down by CID MS/MS of ion corresponding to the peak number 5 at 10.88 min [8],...

The use and development of high-resolving separation techniques as well as highly accurate mass spectrometers is nowadays essential to solve the proteome complexity. Currently, more than a single electrophoretic or chromatographic step is used to separate the thousands of proteins found in a biological sample. This separation step is followed by analysis of the isolated proteins (or peptides) by mass spectrometry (MS) via the so-called soft ionization techniques, such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) combined with the everyday more powerful mass spectrometers. Two fundamental analytical strategies can be employed the bottom-up and the top-down approach. 

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... 

Fig. 1. Native polyacrylamide gel electrophoresis. The protein samples are loaded into the sample wells formed in the top of the gel. An electric field is applied across the gel from top to bottom and the proteins migrate down through the gel. The smaller the protein and the greater its net negative charge, the further it will migrate.

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