Peptide mass spectrometric analysis

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. 

Mass spectrometric analysis of phosphylated peptides after enzymatic digestion of modified cholinesterase. 

Besman M.J. and Shiba D. (1997), Evaluation of genetic stability of recombinant Human Factor VIII by peptide mapping and on-line mass spectrometric analysis. Pharm. Res. 14(8), 1092-1098. 

For routine analysis, it is suggested that mass determination of an HPLC purified product is sufficient to be relatively assured that the correct product has been made. Of course, this approach will neither determine if the correct amino acids have been incorporated into an incorrect sequence nor determine if a substitution of amino acids of identical mass has occurred. However, the numerous checkpoints for automated peptide synthesis (bar codes, printouts, etc.) should greatly reduce the probability of this occurring. Should access to mass spectrometric analysis not be available, amino acid analysis is preferable. 

Some laboratories do not have access to mass spectrometric analysis, but the number is fewer as the cost for this type of instrumentation is decreasing. It is suggested that these laboratories utilize amino acid analysis due to reduced cost and rapid turnaround. Peptide composition and stoichiometry can be determined, the technique is highly reproducible, and can be used to monitor cycle-to-cycle coupling efficiency. However, not all amino acids are recovered quantitatively. Cys and Trp are totally destroyed and must be quantitated using distinctly different hydrolysis procedures. Ser and Thr can be partially destroyed. Some laboratories perform amino acid analysis in addition to mass spectrometric analysis in order to assure peptide composition, stoichiometry, and quantity (see also Sections 7.3, 7.3.1 and 7.3.2). 

Sequencing by both Edman sequence analysis and mass spectrometric analysis can be complimentary and where one may fail in yielding information concerning the synthesis problem, the other may succeed. This is because every peptide has different properties and each technique has advantages and disadvantages. However, sequence analysis is expensive and time consuming, which must be taken into consideration when solving the problems of peptide synthesis, especially those of routine peptide synthesis. 

A large number of wanted and unwanted products can be generated using solid-phase peptide synthesis. To insure quality peptides, it is important to characterize synthesized products with respect to quantity and purity. An array of analytical techniques is needed for peptide analysis one is not enough. Studies performed by the Peptide Synthesis Research Group have shown improved quality of submitted peptides over eight years and have shown the usefulness and necessity of mass spectrometric analysis of peptides. This enhanced capability to characterize wanted and unwanted products should encourage peptide scien-... 

Separation of the modified peptide from the released glycan may be accomplished using a Cig cartridge and the glycan then isolated from the unretained fraction by acid-catalyzed acetylation and organic/aqueous partition. Tandem mass-spectrometric analysis of the recovered peptide yields sequence information allowing location of the modified amino acid. 

F.-G. Hanisch, M. Jovanovic, and J. Peter-Katalinic, Glycoprotein identification and localization of O-glycosylation sites by mass spectrometric analysis of deglycosylated/alkylaminy-lated peptide fragments, Anal. Biochem., 290 (2001) 47-59. 

The assumption can be made that the more frequently a peptide spectrum is observed, the more abundant the protein is in the sample (91). In the mass-spectrometric analysis of peptides, ions derived from peptides entering... 

The integration of SpC data with mass ion intensity information can provide a more accurate indication of abundance (98). Similarly, methods weighting SpC data with data MSI feature extracted ion intensities improves reliability (99). To absolutely quantify proteins requires integration of the individual peptide spectral ions generated during mass-spectrometric analysis. To do this requires extraction of peptide MS2 spectra to obtain ion intensity and masses. By correlating ion intensity and accurate mass with internal standard spikes... 

Today, mass spectrometry has become one of the most widely used analytical techniques in the life sciences [11,12], The mass spectrometric analysis of different classes of biomolecules is reviewed in this chapter peptides, proteins, nucleic acids, oligosaccharides and lipids. Several applications are detailed for each class. Metabolomics, which is the omics science of metabolism, will also be examined at the end of the chapter. 

The region of the protein containing the mutation can be determined by a proteolytic digestion of this protein followed by mass spectrometric analysis of the resulting peptides. Peptides containing the mutation present the same variation of molecular mass as the intact protein. On the other hand, mutation-free peptides have measured molecular masses in agreement with those calculated for the wild-type protein. 

Schlosser, G., Takats, Z., Vekey, G. et al. (2003) Mass spectrometric analysis of combinatorial peptide libraries derived from the tandem repeat unit of MUC2 mucin. J. Peptide Sci., 9, 361-74. 

W. R. Alley, Y. Mechref, and M. V. Novotny, Use of activated graphitized carbon chips for liquid chromatography/mass spectrometric and tandem mass spectrometric analysis of tryptic peptides, Rapid Commun. Mass Spectrom., 23 (2009) 495-505. 

Gygi et al. claimed an offline SCX fractionation coupled with RP and mass spectrometric analysis to be optimal, as opposed to the in-line SCX-RP biphasic methodology used in MD protein identification technology (MudPlT) [35]. The authors argued that the offline SCX approach provides increased loading capacity, improved resolution, greater flexibility and repeated sample analysis. However, MudPIT s comprehensive analysis of complex peptide mixtures avoids the need for complicated switching valves and minimizes sample loss. 

Phosphorylated peptides are generally suppressed in mass spectrometric analysis because of their low ionization rates. 

The peptide mixture that results from tryptic digestion of a protein mixmre is usually fractionated prior to being submitted to mass spectrometric analysis. The goal of the separation is to reduce the complexity of the peptide mixmre to simplify analysis of peptide mass peaks. The most commonly used method in exosome proteome studies is reverse phase high performance liquid chromatography (Feviier et al. 2004 Pisitkun et al. 2004 Potolicchio et al. 2005 Segura et al. 2005 Faure et al. 2006). For this method, a mixture of peptides in aqueous solution is loaded onto a colurrm packed with... 

Figure 3. MALDI-TOF mass spectrometric analysis of Asp-N peptide 19.4 from 24KD r-HuB61...

By mass spectrometric analysis, Asp-N peptide 19.4 (Figure 3) contained primarily one high mannose structure containing 5 mannose units. Asp-N peptide 20.3 produced mass spectra (Figures 4,5) that are consistent with having high mannose structures with 4, 5, and 8 mannose units. 

Figure 3. Structure of the disulfide bonded peptic peptide of r hMCP-1. The structures of the peptide present in the 46 minute fraction obtained form the peptic digest of r hMCP-1 deduced from N-terminal sequencing and MALDI-TOF mass spectrometric analysis.

ESI Mass Spectrometric Analysis. Detergent containing peptide mixtures were analyzed using either a FisionsA G Platform system or a Vestec 201 at the University of Michigan Protein and Carbohydrate Stracture Facility (7). 

ESI mass spectrometry was performed on approximately 32 pmol of peptide mixture in decylglucopyranoside buffer without success (data not shown). PVDF-bound transferrin was digested in lower concentrations of detergent, and substitution of ammonium bicarbonate for Tris, although these conditions are optimum for ESI mass spectrometric analysis the digestion was not successful as determined by HPLC analysis (7). 

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