Tryptic peptide mass mapping

The study of the proteome of the recombinant adenovirus type 5 vectors demonstrated an important apphcation of separation techniques in combination with MS methods in the drug discovery process. With completely sequenced adenovirus genome available, this approach provides a chemically well-dehned method of characterization of structural proteins of recombinant adenoviral vectors. The information of protein MWs, tryptic peptide mass mapping, and sequence tags of tryptic peptides derived from HPLC/MS resulted in the identification of 17 adenoviral proteins/polypeptides in the purified virion. The rapid and accurate identification of viral proteins from recombinant adenoviruses in this study is significant since it provides direct evidence of the maturation stage of adenoviruses, which is closely related to viral infectivity and efficacy in gene therapy. 

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. 

Methods to measure the structure of biopharmaceuticals include tryptic peptide mapping, HPLC, capillary electrophoresis (CE), mass spectroscopy, and circular dichroism spectra. 

Chang, J. P Kiehl, D. E. Kennington, A. 1997. Separation and characterization of the tryptic peptide mapping of recombinant bovine growth hormone by reversed-phase high-performance liquid chromatography electrospray mass spectrometry. Rapid Commun. Mass Spectrom., 11, 1266-1270. 

During detailed characterization of a tryptic map of recombinant human tissue plasminogen activator (rtPA), a minor peak was resolved whose mass was not consistent with the expected set of tryptic peptides (L. Keyt and S.-L. Wu, unpublished observation). N-terminal sequence analysis of this fraction showed that it had a sequence containing residues 276-296 of rtPA with Lys-277 present as Hyl. We developed a modified amino acid analysis program capable of detecting hydroxylysine at levels down to 0.05 residues/mol to determine the distribution of Hyl in rtPA as well as in some other proteins derived from mammalian cells. 

An important development in high-throughput protein identifieation is the introduction of protein database searching [111]. After separation on ID- or 2D-GE, the proteins were blotted onto a membrane and enzymatically digested after reduction and alkylation. The tryptic peptide mixture is analysed by MALDl-MS to achieve a peptide map or peptide mass fingerprint (PMF). The m/z information of the peptides is used to search the protein database, e.g., the Protein Identification Resource (PIR) database [112-114]. If the mass of just 4-6 tryptic peptides is accurately measured (between 0.1 and 0.01%), a useful database search can be performed. 

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. 

Conventional methods for the study of protein phosphorylation rely on radioactive labelling, 2D-GE protein mapping, and Edman degradation. Early studies in LC-MS characterization of protein phosphorylation involve MS-MS analysis of modified tryptic peptide to determine the phosphorylation site by complementary peptide mapping, e.g., [5-7]. In the LC-MS analysis of tryptic and V8-protease digests of a phosphorylated (ppl9) and nonphosphorylated (pl9) 19-kDa cytosolic protein, two sets of ions with a phosphate-characteristic mass difference of 80 Da were observed. Sequence analysis of the relevant peptides by MS-MS showed that phosphorylation occurs at Ser-25 and Ser-38 [7]. 

Figure 3. Identification of a protein by peptide mass fingerprinting. The protein constituents of pig saiiva were separated by SD-PAGE and a protein band was digested with trypsin. The resuitant tryptic peptides were mass-measured using MALDI-ToF mass spectrometry. The peptides in the mass spectrum were either derived from trypsin self-digestion (T) or were derived from the protein in the gel- Database searching with the masses of these peptides led to an unequivocal identification of the protein as SAL (salivary lipocalin). The inset map shows the theoretical tryptic digestion map of this protein, and underneath are the peptides that were observed. In many instances, smaller peptides were visible as partial digestion products.

MALDI-TOF and MALDI-TOF/TOF are straightforward techniques, and a large number of samples can be analyzed rapidly, particularly for masses in the 500 Da to 5 kDa mass range, e.g., to obtain mass maps (MS) and sequences (MS/ MS) of tryptic peptides that have been spotted on plates robotically. However, the lack of resolution and accuracy are limiting when analyzing proteins in the linear mode. Accordingly, MALDI-TOF cannot be used to assess small changes in the molecular mass of proteins, such as those that are due to phosphorylation or when a small molecule is added to a protein. 

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