Peptide maps of tryptic digests

Peptide maps of tryptic digests of oxidized synthetic RNase A showed the expected 14 components plus a spot at the position of lysine. 

V8, as indicated by two-dimensional peptide maps of tryptic digests (44)- It appears, therefore, that various strains of S. aureus produce a major extracellular nuclease with covalent structures extremely similar to that of nuclease V8. Staphylococcus aureus has been reported, however, to produce several minor extracellular nucleases with different isoionic points and heat stabilities 45)- The covalent structures of these materials have not been studied. 

Figure 5. Peptide maps of tryptic digests of normal and sickle cell hemoglobins. Adapted from Ref. 73. The single peptide difference between the two hemoglobins is indicated by shying and...

Figure 8.6 Miiror plots of UHPLC/MS peptide maps from tryptic digests of an innovator mAb and < biosimilar mAb. Peptide mixture was separated on a 2.1 x 150 mm BEH300 1.7 p,m column at 65°C using a 90-min gradient (1%-40%B). The total ion chromatograms (TIC) obtained by the mass spectrometer (A) and a zoom view of charge-reduced, isotope-deconvoluted UHPLC-MS data from 32-35 min (B) are shown. Reproduced with permission from Xie, H., et al. MAbs. 2010.

Location of the variant peptides through peptide mapping of the tryptic digest of the protein using LC-ESI MS or MALDI... 

FIG. 5. Tryptic peptide maps of the maize-branching enzymes showing the similarity between the pair BEIIa/BEIIb and differences between the pair and BEI. Approximately 1.25 nmol of peptide digest was chromatographed on a reverse-phase column. Figure reprinted with permission from Singh and Preiss (1985). 

Scheurer, S.B., Roesli, C., Neri, D. and Elia, G. (2005) A comparison of different biotinylation reagents, tryptic digestion procedures, and mass spectrometric techniques for 2-D peptide mapping of membrane proteins. Proteomics 5, 3035-3039. 

Peptide mass fingerprinting (PMF) of tryptic digests of both the modified and the tmmodified protein (complementary peptide mapping). By careful comparison of the two spectra, m/z shifts can be found, from which the identity of the modification may be elucidated, as well as the tryptic fragment(s) that are actrrally modified. When the amino-acid sequence of the protein is known (and vahdated), the position of the modification may be known. For example. 

Peptides are not as commonly analyzed by CIEF as are proteins one reason is their lower resolution, another their lower (or lack of) detectability at 280 nm (the wavelength mostly used). The separation of tryptic digests (peptide mapping) of proteins have been performed by using absorption detection at 280 nm and refractive index gradient imaging detection no exact correlations were observed between measured and calculated p7 values [1,5]. Refractive index detection is a universal detection method (i.e., independent of chromophores like tyrosine and tryptophan) but suffers from low sensitivity. Assays of trypsin activity have also been performed with laser-induced fluorescence detection for enhanced sensitivity, with detectability down to picomolal concentrations [5,6]. 

Peptide maps of the tryptic digest of myosin have been performed on thin-layer plates (20 x 20 cm) by successive TLC and electrophoresis. In the first-dimension TLC... 

Peptide maps of the tryptic digest of myosin have been performed on thin-layer plates (20 x 20 cm) by successive TLC and electrophoresis. In the first-dimension TLC with chloroform-methanol-ammonium hydroxide 34% (40 40 20, v/v) as eluent, the time was as long as 60 min. The second-dimension electrophoresis with pyridine-glacial acetic acid-water (1 10 489, v/v) buffer and 980 V, 30 mA current, the time was 1 hr. The peptide mixture is applied in amounts of 0.05 to 0.5 mg per peptide map. " In another paper, electrophoresis was applied in one direction on buffered adsorbents, followed by chromatography in the second dimension. Here, phosphate esters were separated by TLC development twice with n-propanol-ammonium hydroxide-water (60 30 1, v/v), and electrophoresis was carried out in 0.28 M acetate buffer (pH 3.6), 1000 V, 35 mA, and 16 min. 

While simple molecular weight measurements do not provide information on the amino acid sequences of peptides, they have been profoundly useful for verifying sequences which are inferred from the nucleic acid sequences of the genes encoding the peptide. In particular, they have been used to verify the sequences of peptides produced by recombinant techniques or by total chemical synthesis, or to reveal possible post-translational modifications. More specific information, however, can be obtained by comparative mass mapping of tryptic (or other enzymatic) digests. This approach is particularly useful when the molecular-ion mass exceeds the mass range of the plasma desorption technique. 

Matrix-associated laser desorption ionization with a time-of-flight mass analyser (MALDl-ToF) was used to examine the crude tryptic peptide mixture from a number of the proteins, without HPLC separation, to provide a mass map, i.e. a survey of the molecular weights of the peptides generated by the digestion process. 

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