Peptides, detection

Table 5.9 Peptides detected during the LC-electrospray-MS (LC-ESMS) analysis of the endoproteinase Lys-C digest from native cytochrome c". Reprinted from Biochim. Biophys. Acta, 1412, Klarskov, K., Leys, D., Backers, K., Costa, H. S., Santos, H., Gnisez, Y. and Van Beenmen, J. J., Cytochrome c" from the obligate methylotroph Methylophilus methylotrophus, an unexpected homolog of sphaeroides heme protein from the phototroph Rhodobacter sphaeroides", 47-55, Copyright (1999), with permission from Elsevier Science...

Figure 12.5 illustrates a typical problem of analysis of minor components present in a matrix of highly abundant ones. Despite the availability of large LC-MS peak capacity (Table 12.3), the number of peptides detected in a semm/plasma digest does not exceed several hundreds (Kapp et al., 2005). These peptides typically match 30-50 high abundant proteins. We believe that the maj ority of remaining proteins/peptides in the sample are present at concentrations well below the LOD of MS instrument. 

Lashuel, H. A., Petre, B. M., Wall, J., Simon, M., Nowak, R. J., Walz, T., and Lansbury, P. T., Jr. (2002). Alpha-synuclein, especially the Parkinson s disease-associated mutants, forms pore-like annular and tubular protofibrils./. Mol. Biol. 322,1089-1102. LeVine, H. (1993). Thioflavine T interaction with synthetic Alzheimer s disease beta-amyloid peptides Detection of amyloid aggregation in solution. Protein Sci. 2, 404—410. Lin, H., Bhatia, R., and Lai, R. (2001). Amyloid beta protein forms ion channels Implications for Alzheimer s disease pathophysiology. FASEB J. 15, 2433-2444. Lorenzo, A., and Yankner, B. A. (1994). Beta-amyloid neurotoxicity requires fibril formation and is inhibited by Congo red. Proc. Natl. Acad. Sci. USA 91, 12243-12247. Luhrs, T., Ritter, C., Adrian, M., Riek-Loher, D., Bohrmann, B., Dobeli, H., Schubert, D., and Riek, R. (2005). 3D structure of Alzheimer s amyl o id-( be la) (1—12) fibrils. Proc. Natl. Acad. Sci. USA 102, 17342-17347. 

Venne, K., Bonneil, E., Eng, K., and Thibault, P. (2005). Improvement in peptide detection for proteomics analyses using nano LC-MS and high-held asymmetry waveform ion mobility mass spectrometry. Anal. Chem. 77 2176-2186. 

Figure 4 Schematic of ELISA for stem peptide detection.

Siliaed, R., Joenvall, H., Caelquist, M., Mutt, V. (1993). Chemical assay for cyst(e)ine-rich peptides detects a novel intestinal peptide ZF-1, homologous to a single zinc-finger motif. Ear. J. Biochem. 211, 377-380. 

Procedure Material Peptides separation Peptides detection ... 

Fischer, F., Wolters, D., Rogner, M. and Poetsch, A. (2006) Toward the complete membrane proteome high coverage of integral membrane proteins through transmembrane peptide detection. Mol. Cell. Proteomics 5, 444-453. 

LeVine H. Ill Thioflavin-T interaction with syndietic Alzheimers-disease beta amyloid peptides- detection of amyloid aggregation in solution. Protein Sci. 1993 2 404 10. 

Figure 13.9. Affinity capillary electrophoresis-UV-raass spectrometry of a 100-tetrapep-tide library screened for binding to vancomycin (104 fxM in the electrophoresis buffer), (a) The elution of peptides was monitored with UV absorbance during capillary electrophoresis, and the elution time irrieased with increasing affinity for vancomycin. (b) Positive ion electrospray mass spectrum with CID of the Tris adduct of the proton-ated peptide detected at —5 min in the electropherogram shown in a (Reproduced from Ref 52 by permission of the American Chemical ardety.)...

Protein identification in a proteome by tiyptic digestion and RPLC-MS relies on the abihty of trypsin to achieve efficient digestion of the proteins in the nuxture, the ability of RPLC to separate the resulting peptides, and deliver them to nano-ESI-MS for mass analysis. The peptides in a complex mixture widely differ in their physicochemical properties, which in turn influence both LC and nano-ESI-MS performance. Data were compared for experimentally detected and in-silico predicted peptides from three different complex protein mixtmes [38]. The peptides detected actnally form only a small subset of the peptides present. 

New methods use combined HPLC/Mass spectrometry to identify modified amino acids. Purified recombinant human insulin-like growth factor separated into two peaks on reverse phase HPLC (Cl 8 column/acidified water) even though other methods indicated it was completely pure. Plasma desorption mass spectrometry of the individual peptides detected a single methionine sulfoxide molecule that was sufficient to decrease the hydrophobicity of the whole protein significantly. Most of the oxidation occurred when the secreted fusion protein was cleaved with hydroxylamine under not strictly anaerobic conditions, but about 5% occurred during die . coli fermentation. 

Comparative Peptide Detection and Identification using Two-Dimensional Chromatography (SCX/Ci8) on an LC-Chip-MS System... 

---