Bottom-up protein identification

In the early LC-MS-MS studies, CID is performed with low-energy collisions by means of triple-quadrapole instruments. Under these conditions, series of N-terminal b-ions and C-terminal y-ions result from cleavages at the peptide bond and charge retention on either side, although double-charge tryptic peptide ions tend to favour fragmentation towards more abimdant y-ions. From these ladders of sequence ions in the MS-MS spectrum, the amino-acid sequence of the peptide may be derived. This is the bottom-up protein identification approach (Ch. 18.3.1). 

The high resolving power of FT-ICR-MS can readily be exploited in bottom-up protein identification. A nice example is the identification of high-abundant proteins in a tryptic digest of human plasma without any prior separation. The 2745 peaks in the spectmm could be reduced to 1165 isotopic clusters and 669 unique masses, 82 of which matched tryptic fragments of albumin (93% sequence coverage) and 16 others transferrin (41%) [39]. The same group showed that a theoretically predicted retention time of a tryptic peptide can be applied as an additional protein identification tool, next to its accurate mass acquired in LC-FT-ICR-MS. [40-41]... 

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. 

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. 

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-... 

Bottom-Up Identification of unknown proteins by analysis of peptides obtained from... 

The same concept in proteomics studies has technological implications, e.g., which method, sample preparation protocols, and instrumentation will be used. Again, top-down analysis will be based on isolation, analysis, and characterization of an intact protein to reveal its function. Fourier transformed ion cyclotron resonance mass spectrometry (FT-ICR) (Marshall et al., 1998) facilitates such approach in protein identification as a result of random fragmentation of an intact molecule. In contrary, bottom-up approach is based on up-front fragmentation of the protein in question using various proteolytic enzymes with known specificity (Chalmers et al., 2005 Millea et al., 2006). In these experiments, trypsin is most commonly used. An important question that remains is whether more... 

The focus in this text is on the application of LC-MS technology. Three general approaches to proteome mining can be discriminated, which are related to the three general approaches to protein identification bottom-up, top-down, and shotgun. The latter essentially is a bottom-up approach, but is discussed separately. 

The bottom-up approach very much resembles classical protein identification strategies. The proteins in the proteome are first separated by 2D-GE (Ch. 17.3), or in some cases by SCX, size-exclusion (SEC), or affinity (AfC) chromatography. Specific proteins are excised from the gel, blotted, or electroeluted. The protein is digested, and the digest is analysed by LC-MS. The EC separation involves either RPLC with microcapillary or nano-LC columns (Ch. 17.5.2), or 2D-LC with typically SEC or SCX in the first dimension and RPLC in the second (Ch. 17.5.4). Alternatively, the sample may be introduced via either direct-infusion nano-ESl (Ch. 17.2), CE-MS (Ch. 17.5.6), or a microfluidic device coupled to MS (Ch. 17.5.5). 

FT-ICR-MS is a superior tool in bottom-up and shotgun protein identification. This is due to its extreme resolving power and accurate-mass determination capabilities as well as the availability of a variety of pre-ICR-cell or in-lCR-cell... 

There are several chromatography-based methods that have been developed to separate proteins prior to identification by mass spectrometry44 48. The idea is to use at least two orthogonal systems to increase the separation efficiency, e.g. ion exchange followed by hydrophobic interaction. Both top-down and bottom-up approaches have been developed. 

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