RESUMEN
A method is described by which a sequence-dependent peptide fingerprint can be rapidly obtained upon partial hydrolysis of peptides with hydrochloric acid and subsequent analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). When synthetic peptides are treated with 3M HCI for 5 min at 110 degrees C, amino acids are released in turn from the C-terminus or, depending on the peptide, from the N-terminus. Sequence information can be deduced by identifying the amino acid whose mass corresponds to the difference in MW between the major hydrolysis products, beginning from the MW of the starting peptide. A similar pattern exclusively from the C-terminus has been obtained using pentafluoropropionic acid as a hydrolyzing agent (Tsugita et al. Eur. J. Biochem. 206, 691 (1992)), but required longer hydrolysis time and more handling prior to analysis. The technique we have developed could be used to obtain a sequence-dependent 'fingerprint' for a peptide cheaply and rapidly, starting with picomole amounts of peptide, because the hydrolysate can be directly analyzed by MALDI. This methodology might be especially useful for confirming the identity of peptides during peptide mapping.
Asunto(s)
Mapeo Peptídico/métodos , Péptidos/análisis , Secuencia de Aminoácidos , Hidrólisis , Rayos Láser , Espectrometría de Masas , Datos de Secuencia Molecular , Peso MolecularRESUMEN
Mass spectrometric techniques for identification of proteins by "mass fingerprinting" (matching the masses of tryptic peptides from a protein digest to the theoretical peptides in a database) such as matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) are rapidly growing in popularity as the demand for high throughput analysis of the proteome increases. This is due, in part, to the ability to automate the technique and the rapid rate with which mass spectra may be acquired. An important factor in the accuracy of the technique is the number of tryptic peptides that are identified in the various searching algorithms that exist. The greater sequence coverage of the parent protein that is obtained, the higher the level of confidence in the identification that is determined. One impediment to high levels of sequence coverage is the bias of MALDI-TOF mass spectrometry to arginine-containing peptides. Increasing the sensitivity to lysine-containing peptides should increase the sequence coverage obtained. In order to achieve this result we have developed conditions to modify the epsilon-amine group of lysine in tryptic peptides with O-methylisourea. The conditions utilized result in the conversion of lysine to homoarginine with no modification of the amine terminus of the peptides. The sensitivity of MALDI-TOF mass spectrometry detection of peptides was increased dramatically following modification. The modification chemistry may be applied to tryptic peptide mixtures prior to desalting and spotting onto MALDI-TOF plates. This technique will be particularly useful for identifying proteins with a high lysine/arginine ratio.
Asunto(s)
Homoarginina/química , Lisina/química , Mapeo Peptídico/métodos , Péptidos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Secuencia de Aminoácidos , Animales , Apoproteínas/análisis , Apoproteínas/química , Bovinos , Grupo Citocromo c/análisis , Grupo Citocromo c/química , Citocromos c , Compuestos de Metilurea/farmacología , Análisis de Secuencia , Albúmina Sérica Bovina/análisis , Albúmina Sérica Bovina/químicaRESUMEN
Hypertrophy is an adaptive response of the heart to myocardial injury or hemodynamic overload that may progress and contribute to cardiac decompensation and eventually to heart failure. The signaling pathways controlling this response in the cardiac myocyte are poorly understood. A data mining effort of a human failed heart cDNA library was undertaken in an effort to identify novel signaling molecules involved in cardiac hypertrophy. This effort identified a novel kinase (MLK7) homologous to the mixed lineage kinase family of proteins. The mixed lineage kinases are mitogen-activated protein kinase kinase kinases (MAPKKKs) which activate stress activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p38 kinase pathways. They contain a catalytic domain with homology to both serine/threonine and tyrosine-specific kinases and a dual leucine zipper. MLK7 is identical to leucine zipper and sterile-alpha motif protein kinase (ZAK) through the leucine zipper domain but has a completely divergent COOH-terminus and shares approximately 40% homology with the other MLKs overall. Expression of MLK7 mRNA is most abundant in skeletal muscle and heart, with expression restricted to the cardiac myocyte. The recombinant histidine tagged MLK7 expressed and purified from insect cells exhibited serine/threonine kinase activity in vitro with myelin basic protein as substrate. When expressed in cardiac myocytes, MLK7 activated SAPK/JNK1, and ERK and p38 to a lesser extent. Additionally, MLK7 altered fetal gene expression and increased protein synthesis in cardiac myocytes. These data suggest that MLK7 is a new member of the mixed lineage kinase family that modulates cardiac SAPK/JNK pathway and may play a role in cardiac hypertrophy and progression to heart failure.
Asunto(s)
Quinasas Quinasa Quinasa PAM/metabolismo , Proteínas Musculares , Miocardio/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Adulto , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Cardiomegalia/enzimología , Cardiomegalia/metabolismo , Células Cultivadas , Clonación Molecular , ADN Complementario , Femenino , Regulación de la Expresión Génica , Biblioteca de Genes , Corazón/fisiología , Humanos , Quinasas Quinasa Quinasa PAM/química , Quinasas Quinasa Quinasa PAM/genética , Sistema de Señalización de MAP Quinasas/fisiología , Datos de Secuencia Molecular , Músculo Esquelético/enzimología , Miocardio/citología , Miocardio/metabolismo , Fosforilación , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Ratas , Proteínas Recombinantes/metabolismo , Distribución TisularRESUMEN
We have previously shown that rat pancreatic islets contain a predominant 135 kDa O-glycosylated protein (p135) that is recognized by immunoprecipitation and Western blotting with anti-O-GlcNAc antibody. In this paper, we show that p135 is also detectable in other rat tissues including brain, heart, liver, spleen, and lung, but not kidney. To identify p135, the protein was purified from rat brain using a multistep procedure including selective absorption with anti-O-GlcNAc antibody. After electrophoresis, and Coomassie staining, the protein was excised from the gel for tryptic digestion. Next, O-methylisourea was used to convert lysine residues to homoarginine to increase the sequence coverage, and MALDI-TOF mass spectrometry detection was performed. MALDI-TOF identified p135 as rat O-GlcNAc transferase (OGT), an identity confirmed by LC/MS of individual peptides. The identification of p135 as OGT is consistent with previous reports of the tissue distribution of OGT, as well as reports that OGT is itself O-glycosylated.