Tryptic peptide analysis proteins

The soluble peak, B, runs exactly coincident with the large subunit of Fraction I protein (Fig. 4). The identity of this peak with the large subunit has been confirmed by tryptic-peptide analysis, and this is the first definitive identification of a protein that is synthesized by chloroplast ribosomes. It is this author s opinion that chloroplast ribosomes represent a high proportion of the cellular ribosomal complement, not because they make a wide range of different proteins, but because one of their products—the large subunit of Fraction I protein—is a component of one of the most abundant proteins. The reason for the abundance of this protein is its very low efficiency as a catalyst for the ribulose-diphosphate... 

DEAE 25W were operated in tandem to map the peptides generated on tryptic hydrolysis of very large proteins, such as albumin genetic variants.197 A useful review that covers many aspects of peptide analysis is available.198... 

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. 

All samples were digested with trypsin and analyzed by cIEF in the first dimension followed by LC-MS/MS as described above. Samples were analyzed in duplicate. Sequence searching was performed using OMSSA. Analysis of the soluble fraction yielded a total of 2856 identified proteins, while the insoluble fraction yielded 3227 proteins. Combined, the fresh-frozen sample yielded 3902 protein identifications. The FFPE portion yielded 2845 protein identifications from 14,178 distinct tryptic peptide sequences, on a par with the fresh-frozen soluble fraction. Combining all identifications gave 4145 proteins. While, the soluble fraction and the FFPE extraction yielded similar numbers of protein identification, both found 25% of their respective protein set uniquely (Fig. 20.4). 

Brancia, F.L., Oliver, S.G., and Gaskell, S.J. (2000) Improved matrix-assisted laser desorption/ionization mass spectrometric analysis of tryptic hydrolysates of proteins following guanidination of lysine-containing peptides. Rapid Comm. Mass Spectrom. 14, 2070-2073. 

Giusti, P., Schaumloffel, D., Encinar, J. R., and Szpunar, J., Interfacing reversed-phase nanoHPLC with ICP-MS and on-line isotope dilution analysis for the accurate quantification of selenium-containing peptides in protein tryptic digests. Journal of Analytical Atomic Spectrometry 20(10), 1101-1107, 2005. 

The best analysis can be achieved when the complexity of a protein mixture is reduced. This will increase the capacity of the MS instrument for analysis. Considering those with approximately 6,000,000 tryptic peptides, it becomes highly challenging unless mixtures are separated to their components (single proteins or peptides) prior to analysis. 

The most obvious effect of a deficiency in vitamin K in animals is delayed blood clotting, which has been traced to a decrease in the activity of prothrombin and of clotting factors VII, IX, and X (Chapter 12, Fig. 12-17). Prothrombin formed by the liver in the absence of vitamin K lacks the ability to chelate calcium ions essential for the binding of prothrombin to phospholipids and to its activation to thrombin. The structural differences between this abnormal protein and the normal prothrombin have been pinpointed at the N terminus of the 560 residue glycoprotein.e f Tryptic peptides from the N termini differed in electrophoretic mobility. As detailed in Chapter 12, ten residues within the first 33, which were identified as glutamate residues by the sequence analysis on normal prothrombin, are actually y-carboxyglutamate (Gla). The same amino acid is present near the N termini of clotting factors VII, IX, and X. 

Determination of the molecular weight difference between the wild- type and the variant TTR obtained by ESI MS limited postulated amino acid substitutions to a relatively small number. Analysis of the tryptic digest of the protein by LC-ESI MS was used to locate more precisely the site of the mutation. Comparison of the tryptic map obtained for the variant with that of a normal sample facilitated detection of the modified tryptic peptide. MS-MS was carried out on the variant tryptic peptide contained in the appropriate chromatographic fractions collected during the LC-ESI MS experiment. 

Inspection of the UV chromatogram from the LC-ESI MS analysis of the tryptic digest of the protein revealed a peak not present in the digestion of wild-type TTR, at retention time 25.95 min. The [M + H]+ of this variant peptide (m/z 1392.6) was consistent with a 27 Da increment to the normal tryptic peptide T4, 22GSPAINVAVHVFR34. The location of the mutation was confirmed by MALDI MS analysis of the chromatographic fraction collected between retention times 25.0-27.5 min. In the MALDI mass spectrum, peaks at m/z 1365.2 (wild-type T4) and m/z 1392.1 (variant T4) are both present (Fig. 8). This observation confirmed that the mass difference between the variant and the wild type peptides is indeed +27 Da. The only possible amino acid substitution that would give rise to a +27 Da shift in that peptide is Ser23 —> Asn. 

N-alkyl silica-based stationary phases typically employed in HPLC separation of peptides and proteins have been investigated to a large extent in CEC mode. In most cases, gradient elution was required to optimize the resolution and analysis time. Tryptic digests maps of cytochrome c by pressurized CEC were better resolved than by pHPLC [93], The experiments were conducted at low pH in the presence of trifluoroacetic acid (TFA) to prevent tailing of basic peptides, on a column packed with 1,5-pm C18 silica particles. It has been... 

Immunochemical studies, tryptic peptide mapping, and end-group analysis suggested that the toxin and agglutinin may have one common and one unique type of subunit each.146,147,648,658,660-662 Thus, a comparison of tryptic peptide maps gave146 a ratio of identical to unique peptides of 1 1. Furthermore, antisera to either the toxin or the agglutinin cross-reacted with the other protein.146,147,658,660,662... 

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