Peptide, amino acid sequence phosphorylated

The amino acid sequence around the serine that is phosphorylated in the presence of inorganic phosphate at low pH can be seen in Table III (55-57). The sequence of Schwartz et al. (55) accounted for 56% of the peptides that contained 32P (20% or more of the peptides were excluded as extreme fractions when the peaks were pooled). The sequence, as far as it is known, is the same for alkaline phosphatase from a mammalian source (58). It is interesting to note, as pointed out by Boyer and others (59-64), that many hydrolytic enzymes with a serine residue at their active site have the same general sequence, i.e., Asp (Glu)-Ser-Ala (Gly). 

Homologues of the mammalian insulin receptor, IR, are the DAF-2 receptor in Caenorhabditis elegans and the IR-like receptor in Drosophila. TTie DAF-2 receptor shares 35% of its amino-acid sequence with the human insulin receptor and 34% with the insulin-like growth factor receptor-1. The tyrosine kinase domain of the DAF-2 receptor is 70% similar and 50% identical to the tyrosine kinase domain of the human insulin receptor. A ligand for DAF-2 has not yet been identified, but an insulin-like peptide is anticipated. Since a t5T>ical insulin receptor substrate, like lRS-1 or IRS-2, has not been found in C. elegans, it is assumed that a COOH-terminal extension of the DAF-2 receptor serves as a built-in receptor substrate, which when phosphorylated, helps to recruit signalling proteins. 

The basic goal of the mass spectrometry measurement in the context of peptide analysis in proteomics and phosphoproteomics is to determine specific attributes that are then used in subsequent database searches to provide 1. the identity of the proteins present in the sample 2. location of the site(s) of phosphorylation in these proteins. Both pieces of information are derived from the mass of the peptide and, most importantly, from the gas-phase dissociation patterns that are diagnostic of the peptide s amino acid sequence and phosphosite location. The gas-phase dissociation patterns are obtained via tandem mass spectrometry (MS/MS). On a phosphoproteome-wide scale, the analysis includes measurement of the attributes for many thousands of individual peptides. 

Most of the samples we have analyzed are unsuitable for normal amino acid sequencing because of the low amounts of peptide ( 1 pmole) and the likelihood of multiple peptides being present. Thus we have not attempted to use extraction with trifiuoroacetic acid in combination with amino acid uencing as done by Meyer et al. (2). In cases where amino acid sequence data is needed, we have used some sample to detect the phosphorylation site and the remainder for standard amino acid sequencing. This protocol avoids having to test and optimize a third sequencing cycle which attempts to identify amino acids and collect radioactivity. 

In the case of the ESTMS-MS data, actual amino acid sequence can be deduced. This is possible due to the CID processes, which breaks the peptides further into amino acid ions. Each amino acid ion has a specific mass and by calculating masses of specific amino acid from the MS spectra, the exact sequence of the peptide and in turn the protein can be deduced. The workhorse of such analysis is a program called Sequest . Since the ESTMS-MS analysis provides information about the actual amino acid sequence, it is also useful to obtain information about protein modifications (such as phosphorylation) and toxicant-induced protein adducts. This has become even easier with the advent of new software tools and highly intelligent algorithms such as SALSA . 

We synthesized lOmer and 15mer peptides, which included the amino acid sequence of the phosphorylated site, ArgArgAlaSerLeu, of PKA. Ser is phosphorylated by PKA in the cell. EDANS-modified lOmer and 15mer peptides were synthesized on bead. After cleavage and deprotection of peptide side chain, dabcyl group was attached at the N-terminal a-amino group. Ser-phosphorylated sensor peptides were also synthesized. The synthesized sensor peptides were purified using HPLC and identified by MALDI-TOF MS (Table 1). 

MBP occurs on specific threonine residues that are proline-directed that is, the phosphorylated amino acids are immediately followed by proline in the amino acid sequence of the protein. In studies of peptide substrates phosphorylated by MAPK it appears that the sequence Ser/Thr-Pro is a minimum consensus phosphorylation sequence for MAPK. However, the amino acids carboxyl- and amino-terminal to this sequence modify the ability of MAPK to covalently attach phosphate. In particular, the placement of proline at position -2 (relative to the phosphorylated amino acid) increases kinase activity. On the basis of these data, the optimal consensus phosphorylation sequence for MAPK is Pro-X-(Ser/Thr)-Pro (Clark-Lewis et al., 1991). Certain proteins, including caldesmon, are phosphorylated by MAPK on sites that do not match this optimal consensus sequence exactly (Adam and Hathaway, 1993). 

A novel phosphorylated peptide has been isolated from the A. suum PFK, after phosphorylation with the mammalian cAMP-dependent protein kinase (28). Its sequence, Ala-Lys-Gly-Arg-Ser-Asp-SerP-Ile-Val-Pro-Thr, is unlike the phosphorylation sequence found in rabbit muscle PFK. The predicted amino acid sequence of H. contortus PFK exhibits about 70% similarity with mammalian PFKs but no sequence is present at the carboxy-terminus which corresponds to the consensus cAMP-dependent phosphorylation site identihed in mammalian PFKs (29). However, although not noted by the authors, H. contortus PFK does have a sequence near its amino-terminus that contains 7 of the 11 amino acids in the A. suum phosphorylated peptide. The role of this phosphorylation site in the regulation of PFK activity requires further study. 

---