Isolating active site peptides has been simplified by recent advances in photolabeling and purification techniques. Previous technical limitations involved: a) low photoinsertion yield and b) lability of the photoinserted bond to reverse phase HPLC conditions. These issues have been favorably addressed with current techniques. Several procedures have been reported to increase the photoinsertion yield. A procedure using immobilized metal affinity chromatography (IMAC) prior to RP-HPLC and reduced HPLC flow rate to lower pressure has helped to overcome the latter limitation. Reduced flow rates (0.5 ml/min compared to 1.0 ml/min) during RP-HPLC purification has been shown to retain close to 80% more label with the photolabeled peptide. This methodology has facilitated the isolation of active site peptides of several nucleotide binding proteins. The steps involved in IMAC are shown in the illustration below.
IMAC provides a mild and rapid pre-purification step prior to HPLC. The sample eluted in the final step of IMAC is enriched several fold with the photolabeled peptide. Subsequent RP-HPLC purification at a lower flow rate provides further purification of photolabeled peptide for amino acid sequence determination. In most cases the site of modification can be identified.
Affinity Selection Techniques for Proteomic Studies
PharmaGenomics, Jul 1, 2003 Surveyed here are methods and applications for isotop-coded cysteine affinity tags, histidine affinity selection, glycosylation affinity selection and affinity selection based upon phosphate groups. Affinity Selection Based Upon Phosphate Groups Phosphorylation is one of the most frequent and important post-translational modifications of proteins. Approximately one-third of all mammalian cellular proteins are phosphorylated. Phosphorylation of serine accounts for roughly 90% of these modifications, with phosphorylation of threonine and tyrosine residues accounting for 10% and 0.1% of the total, respectively. Reversible phosphorylation is known to be involved in the regulation of cellular events and processes, including signal transduction, gene expression and metabolism, and cell growth, division and differentiation. Knowledge of the sites of protein phosphorylation and the correlation of phosphate residency with metabolic changes will be important for analysts’ understanding of cellular regulation. An obstacle in determining the state of protein phosphorylation is the huge complexity of the proteome and the occurrence of potentially important phosphoproteins at low concentrations in the cell. Affinity enrichment of phosphoproteins serves to eliminate interferences and to increase signal in protein identification experiments, which is particularly important in the case of polypeptides phosphorylated at multiple sites because they exhibit signal suppression in sequencing experiments using positive-ion electrospray ionization MS-MS. Analysts have used two affinity approaches for phosphoprotein enrichment. The first is chromatography on immobilized metal affinity chromatography columns complexed with Fe3+, Ga3+ or other metals (14-18). Protein products are loaded onto the immobilized metal affinity chromatography column under acidic conditions (pH 2.5-3.5), unbound nonphosphopeptides are removed from the column with an acidic wash solution and variety of metals, including Fe3+, Ga3+, Al3+ and Zr3+, for immobilized metal affinity chromatography enrichment of phosphopeptides. The best selectivity and recovery was observed with iminodiacetate columns complexed with Ga3+. They found increased binding of nonphosphopeptides when loading was performed outside the pH 2.0-3.5 range. Stensballe and colleagues (17) reported that excessive loading of peptide samples increased the nonspecific binding of nonphosphopeptides (17). In another study, Zhou and co-workers (16) evaluated Fe3+ - and Ga3+ - complexed nitrilotriacetate immobilized metal affinity chromatography materials for direct ionization of phosphopeptides in matrix-assisted laser-desorption inonization. 14. L.M. Nuwaysir and J.T. Stults, J. Am. Soc. Mass Spectrom. 4, 662669 (1993). 15. M.C. Posewitz and P. Tempst, Immobilized gallium(III) affinity chromatography of phosphopeptides. Anal Chem. 1999 Jul 15;71(14):2883-92. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10424175&dopt=Abstract 16. W. Zhou, B.A. Merrick, M.G. Khaledi and K.B. Tomer, Detection and sequencing of phosphopeptides affinity bound to immobilized metal ion beads by matrix-assisted laser desorption/ionization mass spectrometry. J Am Soc Mass Spectrom. 2000 Apr;11(4):273-82. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10757163&dopt=Abstract 17. A. Stensballe, S. Andersen and O.N. Jensen, Characterization of phosphoproteins from electrophoretic gels by nanoscale Fe(III) affinity chromatography with off-line mass spectrometry analysis. Proteomics. 2001 Feb;1(2):207-22.. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11680868&dopt=Abstract
REFERENCES: Biochemistry 1992 May 12;31(18):4479-87. (1581304) Identification of peptides from the adenine binding domains of ATP and AMP in adenylate kinase: isolation of photoaffinity-labeled peptides by metal chelate chromatography. Salvucci ME, Chavan AJ, Haley BE. United States Department of Agriculture, University of Kentucky, Lexington. Photoaffinity labeling with azidoadenine nucleotides was used to identify peptides from the ATP and AMP binding domains on chicken muscle adenylate kinase. Competition binding studies and enzyme assays showed that the 8-azido analogues of Ap4A and ATP modified only the MgATP2- site of adenylate kinase, whereas the 2-azido analogue of ADP modified the enzyme at both the ATP and AMP sites. The positions of the two nucleotide binding sites on the enzyme were deduced by isolating and sequencing the modified peptides. Photolabeled peptides were isolated by a new procedure that used metal chelate chromatography to affinity purify the photolabeled peptides prior to final purification by reverse-phase HPLC. The sequences of the peptides that were photolabeled with the 8-azido analogues corresponded to residues K28-L44, T153-K166, and T125-E135 of the chicken muscle enzyme. The residues that were present in both tryptic- and Staphylococcus aureus V-8 protease-generated versions of these peptides were assigned to the ATP binding domain on the basis of selective photoaffinity labeling with the 8-azidoadenine analogues. These peptides and an additional peptide corresponding to positions I110-K123 were photolabeled with 2-N3ADP. Since I110-K123 was photolabeled by 2-N3ADP but not by 8-N3Ap4A, it was assigned to the AMP binding domain. PMID: 1581304 [PubMed - indexed for MEDLINE]
Biochemistry 1993 Feb 23;32(7):1883-90. (8439545) Identification of a guanine binding domain peptide of the GTP binding site of glutamate dehydrogenase: isolation with metal-chelate affinity chromatography. Shoemaker MT, Haley BE. Division of Medicinal Chemistry & Pharmaceutics, College of Pharmacy, University of Kentucky Medical Center, Lexington 40536-0093. Photoaffinity labeling with [alpha-32P]8N3GTP and [gamma-32P]8N3GTP was used to identify the guanine binding domain of the GTP regulatory site within glutamate dehydrogenase (GDH). Without photolysis, 8N3GTP mimicked the regulatory properties of GTP on GDH activity with 8N3GTP exhibiting a Ki of 5 microM while the Ki for GTP was about 0.6 microM. Under optimal photolabeling conditions saturation of photoinsertion with 1 microgram of GDH revealed an apparent Kd of 9 +/- 4 microM for [gamma-32P]8N3GTP. Photolabeling with this analog could be competitively inhibited with GTP with an apparent Kd of 12 +/- 2 microM. Other nucleotides such as ATP and NAD(P)H could not reduce the amount of photoinsertion as effectively as GTP. ADP could decrease photoinsertion, but only at much higher concentrations. NAD(P)+, GDP, AMP, and GMP had little effect on photoinsertion. Divalent cations Mg2+ and Ca2+ also reduced photoinsertion significantly while the monovalent K+ and Na+ ions had no effect. Aluminum(III)-chelate or iron(III)-chelate affinity chromatography and reversed-phase HPLC were used to purify photolabel-containing peptides generated with either trypsin or chymotrypsin. This identified a portion of the guanine binding domain within the GTP regulatory site as the region containing the sequence Ile439 to Tyr454. Photolabeling of this peptide was prevented 91% by the presence of 300 microM GTP during photolysis. Lys445 was not identified in sequence analyses of the photolabeled peptides. Also, trypsin was unable to cleave the photolabeled peptide at this site. These results suggest that Lys445 may be the residue modified by [alpha-32P]8N3GTP. PMID: 8439545 [PubMed - indexed for MEDLINE]
Biochemistry 1996 Oct 22;35(42):13501-10. (8885829) Identification of adenine binding domain peptides of the NADP+ active site within porcine heart NADP(+)-dependent isocitrate dehydrogenase. Sankaran B, Chavan AJ, Haley BE. Division of Medicinal Chemistry and Pharmaceutics, College of Pharmacy, University of Kentucky Medical Center, Lexington 40536-0082, USA. Photoaffinity labeling with [2'-32P]2N3NADP+ and [32P]2N3NAD+ was used to identify two overlapping tryptic and chymotryptic generated peptides within the adenine binding domain of NADP(+)-dependent isocitrate dehydrogenase (IDH). Photolysis was required for insertion of radiolabel, and prior photolysis of photoprobes before addition of IDH prevented insertion. Photoincorportion of 2N3NAD+ inhibited the enzymatic activity of IDH. Photolabeling of IDH with both [32P]2N3NAD+ and [2'-32P]2N3-NADP+ showed saturation effects with apparent Kds of 20 and 14 microM (+/-12%), respectively. The efficiency of photoincorporation at saturation of binding sites was determined to be about 50%. Also, photolabeling was observed with [32P]8N3ATP and [32P]2N3ATP but with saturation effects observed at lower affinity. With all radiolabeled probes reduction of photoinsertion was effected best by the addition of NADP+ followed by NAD+ and then ATP, indicating that photoinsertion with all the probes was within the NADP+ binding site. Isolation of [32P]2N3NAD+ and [2'-32P]2N3NADP+ photolabeled peptides by use of immobilized boronate and immobilized Al3+ chromatography, respectively, followed by HPLC purification resulted in the identification of overlapping peptides corresponding to Ile244-Arg249 and Leu121-Arg133 (tryptic fragments) and Lys243-His248 and Leu121-His135 (chymotryptic fragments). Trp125 and Trp245 were identified as the sites of photoinsertion based on these residues not being detectable on sequencing, the lack of chymotryptic cleavage at these residues, and the decreased rate of trypsin digestion at nearby Lys243 and Lys127. Sequence analysis of [32P]8N3ATP and [32P]2N3ATP photolabeled peptides gave essentially the same peptide regions being photolabeled but at much lower efficiency, indicating that the effects of ATP on IDH activity are dependent on competition for the same site. PMID: 8885829 [PubMed - indexed for MEDLINE]
Additional References: Photoaffinity labeling of the ATP binding domain of Rubisco activase and a separate domain involved in the activation of ribulose-1,5-bisphosphate carboxylase/oxygenase. Salvucci ME, Chavan AJ, Klein RR, Rajagopolan K, Haley BE. Biochemistry. 1994 Dec 13;33(49):14879-86. (7993914) Photoaffinity labeling of creatine kinase with 2-azido- and 8-azidoadenosine triphosphate: identification of two peptides from the ATP-binding domain. Olcott et. al., (1994). Biochemistry 33:11935-11941.(7918412) Identification of peptides within the base binding domains of the GTP- and ATP-specific binding sites of tubulin. Jayaram and Haley (1994). J. Biol. Chem. 269:3233-3242.(8106359) |
