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 1. Reverse-phase chromatographic maps of tryptic digests of reduced caiboxymethylated ribonuclease preparations (a) RNase A control (b) RNase S control and (c) RNase S treated for 1 min with C2N2. Modified and unmodified protein samples were analyzed by reverse-phase HPLC (Perkin-Elmer 250 Binary Pumping Model 235, Diode array Detector, Vydac C-18, 18TP54 Column) using the method of McWherter et al. (18). The sample digests were frozen and stored at -70°C until analyzed.

Alkylammonium acetate buffer systems have also attracted limited attention for the separation of tryptic digests of proteins on octadecyl silica 60). The ammonium formate or trifluoroacetate buffers have also been used in the reversed-phase HPLC separation of the tryptic, chymotryptic, and carboxypeptidase A digests 136) of a-melanotropin and its N,0-diacetylserine analog as well as for enzymatic digests of the adrenocorticotropic family 137). Ammonium bicarbonate eluent systems have proved useful in mapping studies due to the eluent s volatility and differ-... 

A fully carbamylated derivative of rabbit muscle phosphoglucose isomerase was utilized by James and Noltmann (1973), both for a quantitative amino-terminal analysis and for tryptic mapping experiments. Tryptic digestion of the derivative was restricted entirely to the arginyl residues. 

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]. 

Originally, we entertained the possibility that YPg and 7P4 were related proteins (11). However, preliminary results of tryptic digests of VPg and 4 indicate that these viral polypeptides have no overlapping amino acid sequences (19) Experiments are now in progress to map VPg within the viral genome. 

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. 

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.

Peptide mapping (the separation of the tryptic digest) of /i-lactoglobulin and human growth hormone (hGH). 

An ACE-MS hyphenation was utilized for the linear epitope mapping (17). The tryptic digest of /8-endorphin was mixed with an anti-/8-endorphin antibody and subsequently analyzed by ACE-ESI-MS. The procedure requires only femtomole amounts of antibody and peptide digest. More technical details of the method are described in Chapter 13 of this book. 

Fig. 4 Epitope mapping by ACE/MS in the positive ESI mode. (A) Tryptic digest, 4.6 pmol//j,E, 10-s pressure injection (B) tryptic digest, 10-s injection followed by 25-s injection of antibody (4.7 pmol//xL). Selected ion electropherograms for the m/z. indicated and the total ion electropherograms, respectively. It is obvious that peptide 1 (Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys) is captured by the antibody. See text for further explanation. (Reprinted with permission from Ref. 40. Copyright 1997 American Chemical Society.)... 

Ballihaut, G., Tastet, L., Pecheyran, C., Bouyssiere, B., Donard, O., Grimaud, R., and Lobinski, R., Biosynthesis, purification and analysis of selenomethionyl calmodulin by gel electrophoresis-laser ablation-ICP-MS and capillary HPLC-ICP-MS peptide mapping following in-gel tryptic digestion. Journal of Analytical Atomic Spectrometry 20(6), 493 99, 2005. 

According to a recent report (55), /3-lactamase I of strain 569 can be further resolved by P-cellulose chromatography. The three fractions thus obtained appear to retain their identity on gel electrophoresis, even in the presence of urea, and yield distinctive tryptic digest maps. 

Nuclease behaves like a typical globular protein in aqueous solution when examined by classic hydrodynamic methods (40) or by measurements of rotational relaxation times for the dimethylaminonaphth-alene sulfonyl derivative (48)- Its intrinsic viscosity, approximately 0.025 dl/g is also consistent with such a conformation. Measurements of its optical rotatory properties, either by estimation of the Moffitt parameter b , or the mean residue rotation at 233 nin, indicate that approximately 15-18% of the polypeptide backbone is in the -helical conformation (47, 48). A similar value is calculated from circular dichroism measurements (48). These estimations agree very closely with the amount of helix actually observed in the electron density map of nuclease, which is discussed in Chapter 7 by Cotton and Hazen, this volume, and Arnone et al. (49). One can state with some assurance, therefore, that the structure of the average molecule of nuclease in neutral, aqueous solution is at least grossly similar to that in the crystalline state. As will be discussed below, this similarity extends to the unique sensitivity to tryptic digestion of a region of the sequence in the presence of ligands (47, 48), which can easily be seen in the solid state as a rather anomalous protrusion from the body of the molecule (19, 49). 

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