Protein mass

Snyder, A.P., Interpreting Protein Mass Spectra A Comprehensive Resource, American Chemical Society, Washington, D.C., 2000. 

Silk fibers, which have incredible strength, comprise well-ordered microcrystals of P-sheets that make up about 30% of the protein mass, interspersed in a matrix of polypeptide chains without order. The p strands of the sheets are oriented parallel to the fiber axis. 

FIG. 3 Three-dimensional model of the protein mass distribution of the S-layer of Bacillus stearothermophilus NRS 2004/3a [(a) outer, (b) inner face]. The square S-layer is about 8 nm thick and exhibits a center-to-center spacing of the morphological units of 13.5 nm. The protein meshwork composed of a single protein species shows one square-shaped, two elongated, and four small pores per morphological unit. (Modified from Ref. 7.)... 

FIGURE 3.2 (A) MALDI TOF protein mass spectral barcodes of the 16 honeys of known... 

The use of CIEF in combination with FTICR has been demonstrated in an analysis of the E. coli proteome (Jensen et al., 1999). For these experiments, E. coli was grown in a medium depleted of rare isotopes in order to increase the mass measurement accuracy. The high abundance isotopes are present at approximately 98.89% 12C, 99.63% 14N and 99.985% H. For peptides, the presence of rare isotopes does not significantly change the spectra but with undigested proteins, mass accuracy can be limited by the broadened distribution of ions of any given protein due to the incorporation... 

Encouraged by this spectral reproducibility, we focused our efforts on the particularly challenging problem of distinguishing bacterial strains by MALDI MS. We developed a modified correlation approach22 that relies on two fundamental qualities of bacterial mass spectra. First, because different bacterial strains of the same species have substantial, if not complete, genetic overlap, most of the protein masses observed with two different strains will be identical. This feature limits the value of the biomarker approach that is commonly used to differentiate bacteria species. Second, as just noted, closely controlled sample preparation and mass analysis procedures can result in highly reproducible results.22 The modified correlation approach takes advantage of subtle, yet reproducible, differences in mass spectra obtained from dif-... 

Upon completion of the ProteinTrawler program, the text hie contains a cumulative hst of all the protein masses that were observed upon deconvolution of the individual summed spectra. This text hie records mass, intensity, and retention time. The retention time information is held in the text hie for... 

The feasibility of this approach to not only differentiate pathogenic and nonpathogenic strains of bacteria based on significant differences in protein mass but also on the basis of variations in levels of protein expression was demonstrated using a method for quantitating protein expression by LC/MS of whole proteins.54 This method is based on the fact that some proteins present in cells are abundant universal proteins whose expression levels exhibit little variation. This method demonstrates that these co-extracted proteins can be used as internal standards to which the other proteins in the sample can be compared. By comparing the intensities of a selected protein to a marker protein, or internal standard, a relative ratio is obtained. This ratio... 

Since ProteinTrawler records the retention time of each protein mass, it is a simple endeavor to maintain chromatographic conditions, split the flow that exits the LC column with a small portion set to the mass spectrometer to monitor for assurance that there were no changes in the retention time that would hinder the pooling of fractions from multiple runs, and to facilitate the determination of which fractions contained the desired proteins. In our experimental setup, the flow was split after the column with 25% of the flow going to the mass spectrometer while the remaining diverted to an HP1100 fraction collector. The fraction collector was used to collect fractions at 1.0-minute intervals. 

Vaidyanathan, S. Broadhurst, D. I. Goodacre, R. Kell, D. B. Explanatory optimisation of protein mass pectrometry via genetic search. Anal. Chem. 2003, 75, 6679-6686. 

An algorithm has been developed to search databases for suites of protein masses automatically (Pineda et al.).65 In this case data from only one axis is being used, m/z values for the protonated molecular ions. Relative intensities are not used in the analysis. The algorithm also assesses the probability of accidental matches and false identifications. Significance testing allows a value to be assigned to each microorganism identification. 

Wang, Z. Dunlop, K. Long, S. R. Li, L. Mass spectrometric methods for generation of protein mass database used for bacterial identification. Anal. Chem. 2002, 74,3174-3182. 

Variations on the spectral peaks from different species of the same genus were also observed. Three species of Pseudomonas produced the spectra shown in Figure 14.2. These spectra are clearly unique and were used to correctly identify unknown samples. Because of peak ratio reproducibility issues in bacterial protein profiles obtained by MALDI MS,11 a fingerprint approach that had been used for other mass spectrometry approaches has not been used. The profile reproducibility problem was first recognized by Reilly et al.12,13 and later researched by others in the field.14,15 As a later alternative, a direct comparison of the mass-to-charge ratio (m/z) of the unknown mass spectral peaks with a database of known protein masses has been used to identify unknown samples.14... 

Because online separations provide such a wealth of information about target proteins, interpretation becomes of critical importance in order to make full use of the data. The first step in any analysis of LC-MS data involves integration and deconvolution of sample spectra to determine protein mass and intensity. In manual analysis (Hamler et al., 2004), users identify protein umbrellas, create a total ion chromatogram (TIC), integrate the protein peak, and deconvolute the resulting spectrum. Deconvolution of ESI spectra employs a maximum entropy deconvolution algorithm often referred to as MaxEnt (Ferrige et al., 1991). MaxEnt calculates... 

FIGURE 13.5 The total ion chromatogram and deconvoluted protein mass map for a ID LC/MS analysis of yeast ribosomal proteins. The bubble size is proportional to component intensity. 

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). 

Analysis, Fate and Removal of Pharmaceuticals in the Water Cycle Food Contaminants and Residue Analysis Protein Mass Spectrometry... 

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