Identification of short terminal motifs enriched by antibodies using peptide mass fingerprinting.

MOTIVATION: Mass spectrometry-based protein profiling has become a key technology in biomedical research and biomarker discovery. Sample preparation strategies that reduce the complexity of tryptic digests by immunoaffinity substantially increase throughput and sensitivity in proteomic mass spectrometry. The scarce availability of peptide-specific capture antibodies limits these approaches. Recently antibodies directed against short terminal motifs were found to enrich subsets of peptides with identical terminal sequences. This approach holds the promise of a significant gain in efficiency. TXP (Triple X Proteomics) and context-independent motif specific/global proteome survey binders are variants of this concept. Principally the binding motifs of such antibodies have to be elucidated after generating these antibodies. This entails a substantial effort in the lab, as it requires synthetic peptide libraries and numerous mass spectrometry experiments. RESULTS: We present an algorithm for predicting the antibody-binding motif in a mass spectrum obtained from a tryptic digest of a common cell line after immunoprecipitation. The epitope prediction, based on peptide mass fingerprinting, reveals the most enriched terminal epitopes. The tool provides a P-value for each potential epitope, estimated by sampling random spectra from a peptide database. The second algorithm combines the predicted sequences to more complex binding motifs. A comparison with library screenings shows that the predictions made by the novel methods are reliable and reproducible indicators of the binding properties of an antibody.

Degradation of the arginyl-tRNA synthetase protein during purification by affinity chromatography on immobilized total tRNA and immobilized tRNA, specific for arginyl-tRNA synthetase.

Arginyl-tRNA synthetase was prepared from Bom:NMRI mouse liver postribosomal supernatants. Purification was performed by either gel filtration on Sephadex G-200 or chromatofocusing before affinity chromatography on immobilized total tRNA (mixture of tRNA's, specific for all aminoacyl-tRNA synthetases), followed by affinity chromatography on immobilized tRNA, specific for arginyl-tRNA synthetase. The purification factor for arginyl-tRNA synthetase activity was 16700x. Following sodium dodecyl sulphate polyacrylamide electrophoresis several protein bands were found in the purified material. All these protein bands were shown to possess arginyl-tRNA synthetase activity.

Autophosphorylation of degradation products of arginyl-tRNA synthetase protein, isolated from Bom:NMRI mouse liver.

Proteins of M(r) 140, 60, 57, 50, 22, and 14 kDa, all possessing arginyl-tRNA synthetase activity, were subjected to phosphorylation without addition of exogenous protein kinase activity. All proteins except the 14 kDa were shown to contain endogenous protein kinase activity. The product of autophosphorylation of the 60, 57, 50, and 22 kDa proteins was identified as a phosphorylated 14.9 kDa molecule. In some cases higher molecular weight phosphorylated proteins were also detected. Properties of the 60 kDa species are described.

Purification of aminoacyl-tRNA synthetase kinase activities associated with threonyl- and tyrosyl-tRNA synthetases isolated from Bom:NMRI mouse liver.

Aminoacyl-tRNA synthetase kinase activities and aminoacyl-tRNA synthetase activities prepared from postribosomal supernatants of Bom:NMRI mouse liver comigrated through the following purification steps: 1 a) gel filtration on Sephadex G-200, 1 b) chromatofocusing, 2) affinity chromatography on immobilised total tRNA (mixture of tRNA's, specific for all aminoacyl-tRNA synthetases) and 3) affinity chromatography on immobilised tRNA, specific for each of threonyl- and tyrosyl-tRNA synthetases. The purification factors for threonyl- and tyrosyl-tRNA synthetases were about 17000x. The purified synthetases showed only one protein band following sodium dodecyl sulphate polyacrylamide gel electrophoresis. The purified threonyl- and tyrosyl-tRNA synthetase proteins were found also to possess threonyl- and tyrosyl-tRNA synthetase kinase activities, respectively. The purification of tRNA(Thr) and tRNA(Tyr) as well as a method for the renaturation and identification of threonyl- and tyrosyl-tRNA synthetase activities in protein bands obtained by SDS PAGE are described.

Reversal of the effect of 17-beta-estradiol 17-beta-acetate on aminoacyl-tRNA synthetase phosphatase and aminoacyl-tRNA synthetase activities in the uterus and liver of ovariectomized Bom:NMRI mice by 2-deoxyadenosine 3-phosphate.

When an adenylyl cyclase inhibitor, 2-deoxyadenosine 3'-phosphate, was administered together with 17-beta-estradiol 17-beta-acetate on ovariectomized Bom:NMRI mice, the short term effects of the hormone on the uterus aminoacyl-tRNA synthetase phosphatase and aminoacyl-tRNA synthetase activities were reversed, whereas the effect on the liver enzymes was about the same for 17-beta-estradiol 17-beta-acetate and for 2-deoxyadenosine 3-phosphate.

The influence of 17-beta-estradiol 17-beta-acetate on adenylyl cyclase activity and aminoacyl-tRNA synthetase phosphatase activity in ovariectomized NMRI mice.

Adenylyl cyclase activity in the uterus of ovariectomized NMRI mice showed an increase of 87% four minutes after intraperitoneal administration of 17-beta-estradiol 17-beta-acetate in water solution, while in the liver of the same animals activity had decreased by 57%. At the same time, the activity of the 51 kDa form of the aminoacyl-tRNA synthetase phosphatase in the uterus showed an increase of 400%, while in the liver a decrease of 60% was observed. Sixty minutes after hormone injection the adenylyl cyclase activities showed no difference from controls.

The activities of aminoacyl-tRNA synthetase phosphatase and aminoacyl-tRNA synthetases in MPC-11 and Krebs II ascites cells.

The activity of aminoacyl-tRNA synthetase phosphatase as well as the activities of aminoacyl-tRNA synthetases in Krebs II ascites cells and MPC-11 cells have been investigated. The activity of the phosphatase was greater in the tumor cells than in normal tissues. The aminoacyl-tRNA synthetase activities were 100-300 times higher than the activities found in the uterus of ovariectomized mice, but not very different from the activities found in the liver. The influence of cyclic AMP. 2-deoxyadenosine 3-phosphate and 2-deoxyguanosine 3-phosphate on the growth of MPC-11 cells, grown in suspension culture, was also investigated.

Chromatofocusing of aminoacyl-tRNA synthetases, extracted from NMRI mouse liver.

Chromatofocusing of 17 aminoacyl-tRNA synthetases extracted from NMRI mouse liver is described and the apparent isoelectric points of these enzymes are presented. Each of 15 aminoacyl-tRNA synthetases was present in two peaks. Isoleucyl-tRNA synthetase showed only one peak and arginyl-tRNA synthetase was present in three peaks. Phosphorylation/dephosphorylation experiments with arginyl-tRNA synthetase indicate that the peaks represent phosphorylated and unphosphorylated synthetase protein. One example of detection of increased protein phosphorylation during a biological experiment is presented.

Comparison of the rate of aminoacylation of tRNA isolated from NMRI mouse liver with tRNA isolated from Krebs II ascites or mouse plasmacytoma cells.

A comparison of the initial rates of aminoacylation of tRNAs isolated from different sources for 17 amino acids was performed. tRNA was isolated from NMRI mouse liver (tRNA L) and from Krebs II ascites tumors (tRNA Asc), and aminoacyl-tRNA synthetases were prepared from the latter cells. The aminoacylation of tRNA Asc was 31-88% slower than the charging of tRNA L. In similar studies, tRNA from a mouse plasmacytoma tumor (tRNA Mt) and from suspension cultured cells of the same cell line (tRNA M) were compared to tRNA L in the aminoacylation reaction catalysed by synthetases isolated from tumor or suspension cultured cells. About half of the tRNAs (Mt or M) for the 17 amino acids tested differed in charging rate when compared to tRNA L, but the differences were not as great as those observed in the experiments where tRNA Asc and tRNA L were compared.

The role of cyclic 3',5'-AMP in the regulation of aminoacyl-tRNA synthetase activities in mouse uterus and liver following 17beta-oestradiol treatment. Activation of a phosphoaminoacyl-tRNA synthetase phosphatase by phosphorylation with cyclic 3',5'-AMP dependent protein kinase.

The changes in the activities of 17 aminoacyl-tRNA synthetases induced by phosphorylation [1] were reversed by the action of cyclic AMP in preparations from both uterus and liver. Cyclic AMP also inhibited the phosphorylation of aminoacyl-tRNA synthetase protein by endogenous non-cyclic AMP-dependent protein kinase and [gamma-32P]ATP. The effect was not due to a stimulation of phosphoaminoacyl-tRNA synthetase phosphatase or to an influence of cyclic AMP on aminoacyl-tRNA synthetases. The activity of phosphoaminoacyl-tRNA synthetase phosphatase was increased by treatment with endogenous cyclic AMP-dependent protein kinase, ATP and cyclic AMP. Affinity chromatography of the 32P-labeled phosphorylated phosphosynthetase phosphatase protein followed by gel electrophoresis showed that the activated phosphatase was phosphorylated. In the uterus, the changes in 17 aminoacyl-tRNA synthetase activities observed 5 min after dibutyryl cyclic AMP administration to ovariectomized mice were similar to those observed after 17beta-oestradiol treatment, whereas in the liver the changes in these activities were the opposite to those found after treatment with 17beta-oestradiol. A mechanism for the regulation of the 17 aminoacyl-tRNA synthetase activities is proposed, which suggests that the synthetase activities inhibited (group I) or stimulated (group II) by phosphorylation with a non-cyclic AMP-dependent aminoacyl-tRNA synthetase kinase are reactivated (group I) or inhibited (group II), respectively, by the action of a cyclic AMP-dependent phosphatase kinase through the increased activity of phosphorylated phosphoaminoacyl-tRNA synthetase phosphatase.

III. The RNA component of aminoacyl-tRNA synthetase complexes isolated from mouse liver. Absence of amino acid accepting activity.

A method is described which permits the simultaneous isolation and separation of insoluble aminoacyl-tRNA synthetase protein - RNA complexes containing high specific synthetase activity, and soluble tRNA which retains 25% to 50% of its specific amino acid accepting activity. A possible amino acid accepting activity of the RNA part of the insoluble aminoacyl-tRNA synthetase protein - RNA complex was investigated by assaying the unchanged complex and the RNA obtained after dissociation from the protein part of the synthetase complex. No amino acid accepting activity was found.

I. A study of the stages in the quantitative isolation of aminoacyl-tRNA synthetase activities from mouse liver.

The elution profiles of 17 aminoacyl-tRNA synthetases from chromatography of 149 000 x g supernatant on Sephadex G-200 were determined as well as the influence of different methods of homogenization and of chromatography on DEAE-cellulose on the elution profiles. With gentle homogenization all synthetases were eluted in the void volume in four different peaks, containing (a) leucyl- and phenylalanyl-, (b) lysyl-, prolyl-, isoleucyl-, methionyl-, glycl-, and valyl-, (c) arginyl-, alanyl-, and asparaginyl- and (d) aspartyl-, histidyl-, seryl-, threonyl-, glutaminyl-, and tyrosyl- tRNA synthetases. With less gentle homogenization, peaks of lower molecular weight appeared. More than two peaks for each aminoacyl-tRNA synthetases were never found. Of the aminoacyl-tRNA synthetases examined, alanyl-,arginyl-, aspartyl-, leucyl- and lysyl-tRNA synthetases were not inactivated by chromatography on DEAE-cellulose, whereas phenylalanyl- and seryl-tRNA synthetases lost 60% of their activity.

II. The influence of 17-beta-oestradiol on aminoacyl-tRNA synthetases from mouse uterus and mouse liver.

The activities of 17 aminoacyl-tRNA synthetases have been determined in liver and uterus preparations of mice. One group of mice were castrated and seven days later given 5 mug 17-beta-oestradiol in olive oil; a similar dose was given after 24 h. The animals were sacrificed one day later. A control group of mice which were also castrated, received olive oil without 17-beta-oestradiol. As the aminoacyl-tRNA synthetases may be found both in high molecular weight and low molecular weight forms, the forms present in the preparations are discussed. The activities of the aminoacyl-tRNA synthetases from uterus augmented under the influence of 17-beta-oestradiol, but to different degrees. The increase in the activities of isoleucyl- and prolyl-tRNA synthetases was not significant. In liver, only the activity of lysyl-tRNA synthetase augmented significantly.