A guide to mass spectrometry-based quantitative proteomics

Proteomics has become an attractive science in the postgenomic era, given its capacity to identify up to thousands of molecules in a single, complex sample and quantify them in an absolute and/or relative manner. The use of these techniques enables understanding of cellular and molecular mechanisms of diseases and other biological conditions, as well as identification and screening of protein biomarkers. Here we provide a straightforward, up-to-date compilation and comparison of the main quantitation techniques used in comparative proteomics such as in vitro and in vivo stable isotope labeling and label-free techniques. Additionally, this chapter includes common methods for data acquisition in proteomics and some appropriate methods for data processing. This compilation can serve as a reference for scientists who are new to, or already familiar with, quantitative proteomics.191633

Improving metallomics information related to transgenic and non-transgenic soybean seeds using 2D-HPLC-ICP-MS and ESI-MS/MS

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)This work reports the use of 2D-HPLC-ICP-MS to enlarge metallomics information when considering soybean seeds. Separations using size exclusion chromatography (SEC) allowed the identification of three metal fractions: the first corresponding to molecular weights from 38.1 to 181.1 kDa, the second from 8.2 to 17.2 kDa and the third from 0.4 to 3.8 kDa. In a second dimension, using anion exchange chromatography (AEX), three sub-fractions containing Fe, Mg and Mn, one containing Cu, and three containing Co, Cu, Mg, Mn and Zn were obtained. After these separations, 33 proteins were identified using the ESI-MS/MS technique, and divided into four functional categories: plant growth/cell division, protein destination and storage, metabolism and unclassified proteins. Among the identified proteins, proteins previously related to metals were found.44373378Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Analysis of secondary structure in proteins by chemical cross-linking coupled to MS

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Chemical cross-linking is an attractive technique for the study of the structure of protein complexes due to its low sample consumption and short analysis time. Furthermore, distance constraints obtained from the identification of cross-linked peptides by MS can be used to construct and validate protein models. If a sufficient number of distance constraints are obtained, then determining the secondary structure of a protein can allow inference of the protein's fold. In this work, we show how the distance constraints obtained from cross-linking experiments can identify secondary structures within the protein sequence. Molecular modeling of alpha helices and beta sheets reveals that each secondary structure presents different cross-linking possibilities due to the topological distances between reactive residues. Cross-linking experiments performed with amine reactive cross-linkers with model alpha helix containing proteins corroborated the molecular modeling predictions. The cross-linking patterns established here can be extended to other cross-linkers with known lengths for the determination of secondary structures in proteins.1217SI27462752Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Instituto Nacional de Ciencia e Tecnologia em Bioanalitica (INCTBio)Genoprot Program for Intracelular PeptidesFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Protein Succinylation and Malonylation as Potential Biomarkers in Schizophrenia

Two protein post-translational modifications, lysine succinylation and malonylation, are implicated in protein regulation, glycolysis, and energy metabolism. The precursors of these modifications, succinyl-CoA and malonyl-CoA, are key players in central metabolic processes. Both modification profiles have been proven to be responsive to metabolic stimuli, such as hypoxia. As mitochondrial dysfunction and metabolic dysregulation are implicated in schizophrenia and other psychiatric illnesses, these modification profiles have the potential to reveal yet another layer of protein regulation and can furthermore represent targets for biomarkers that are indicative of disease as well as its progression and treatment. In this work, data from shotgun mass spectrometry-based quantitative proteomics were compiled and analyzed to probe the succinylome and malonylome of postmortem brain tissue from patients with schizophrenia against controls and the human oligodendrocyte precursor cell line MO3.13 with the dizocilpine chemical model for schizophrenia, three antipsychotics, and co-treatments. Several changes in the succinylome and malonylome were seen in these comparisons, revealing these modifications to be a largely under-studied yet important form of protein regulation with broad potential applications

Insights into scorpion venom peptides: Alternative processing of beta-KTx propeptide from Tityus serrulatus venom results in a new naturally occurring thimet oligopeptidase inhibitor

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Most functions attributed to Tityus serrulatus venom (TsV) are related to active molecules onion-channels; however, here we describe a new pentapeptide that was discovered through enzymatic assay selection using EP24.15. The primary structure analysis revealed the sequence KEXXG (X means Ile or Leu), similar to the sequence present in the beta-KTX propeptide described from the venom of Tityus spp. We confirmed through HPLC analysis that KEILG is the peptide present in TsV, but that KELLG also inhibits EP24.15 although through different mechanisms. (C) 2012 Elsevier Inc. All rights reserved.403033Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)INCTTOXConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Proteomic analysis of the reproductive tract fluids from tropically-adapted Santa Ines rams

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)The present study is focused on the proteome of reproductive tract fluids from tropically-adapted Santa Ines rams. Seminal plasma, cauda epididymal (CEF) and vesicular gland fluid (VGF) proteins were analyzed by 2-D electrophoresis and mass spectrometry. Seminal plasma maps contained 302 +/- 16 spots, within the 4-7 pH range. From these maps, 73 spots were identified, corresponding to 41 proteins. Ram Seminal Vesicle Proteins (RSVP) 14 and 22 kDa and bodhesins 1 and 2 represented the most abundant seminal components. Other seminal proteins included clusterin, angiotensin-converting enzyme, matrix metalloproteinase-2, tissue-inhibitor of metalloproteinase-2, plasma glutamate carboxypeptidase, albumin, lactoferrin, alpha enolase, peroxiredoxin, leucine aminopeptidase, beta-galactosidase, among others. Later, seminal plasma gels were run within narrow pH intervals (3.9-5.1; 4.7-5.9; 5.5-6.7), allowing the additional identification of 21 proteins not detected in 4-7 pH maps. Major proteins of CEF and VGF were albumin and transferrin, and RSVPs, respectively. Western blots confirmed that RSVPs were mainly present in VGF while bodhesins, in VGF and CEF. Based on RT-PCR, RSVP and bodhesin genes were primarily expressed in the vesicular glands. In summary, the reproductive tract fluids of Brazilian hairy rams contain several categories of proteins, with potential roles in sperm protection, capacitation, acrosome reaction and sperm-oocyte interaction. This article is part of a Special Issue entitled: Farm animal proteomics. (c) 2012 Elsevier B.V. All rights reserved.7514SI44364456Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Ceara State Research Foundation (FUNCAP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Parameters of the Reproductive Tract, Spermatogenesis, Daily Sperm Production and Major Seminal Plasma Proteins of Tropically Adapted Morada Nova Rams

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Contents This study describes the reproductive parameters of Morada Nova rams, a breed of hair sheep from Brazil and with unique adaption to tropical environments. At 42weeks of age, 15 rams were subjected to semen collection and, 1week later, animals were slaughtered for collection of testes, epididymis and accessory sex glands. We conducted 2-D electrophoresis of seminal plasma proteins and major spots of stained gels were identified by LC-MS/MS. Total RNA was isolated from testis, epididymis and vesicular glands and subjected to qPCR. At slaughter, scrotal circumference and testicular weight were 27.5 +/- 0.5cm and 109.5 +/- 6.0g, respectively. Seminiferous tubule (ST) diameter was 188.3 +/- 4.0m and each testis contained 1.9 +/- 0.1 Sertoli cells (x10(9)). Each Sertoli cell supported 0.1 +/- 0.01 A spermatogonia, 3.0 +/- 0.2 pachytene spermatocytes and 7.7 +/- 0.5 round spermatids/tubule cross section. Daily sperm production reached 5.6x10(6)cells/g of testis parenchyma. Testis size appeared as indicative of ST diameter and associated with epididymal measurements, as well as with the population of round spermatids and Sertoli cells/testis. Rams with heavier testes had greater daily sperm production and more Sertoli cells/testis. We detected 90.9 +/- 9.6 spots per 2-D gel of seminal plasma. Major seminal proteins were identified as ram seminal vesicle proteins at 14 and 22kDa, representing 16.2% and 12.8% of the total intensity of valid spots in the gels, respectively. Expression of both genes was greater in the vesicular glands as compared to testis and epididymis. Pixel intensity for those proteins in the 2-D gels was significantly correlated with seminal vesicle weight. This is the first description of the basic reproductive aspects of Morada Nova rams, including protein profiles of their seminal plasma. These findings will allow a better understanding of their reproductive physiology.493409419Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Ceara State Agency for Technology and Scientific Development (FUNCAP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Parameters of the reproductive tract, spermatogenesis, daily sperm production and major seminal plasma proteins of tropically adapted morada nova rams

Contents This study describes the reproductive parameters of Morada Nova rams, a breed of hair sheep from Brazil and with unique adaption to tropical environments. At 42weeks of age, 15 rams were subjected to semen collection and, 1week later, animals were slaughtered for collection of testes, epididymis and accessory sex glands. We conducted 2-D electrophoresis of seminal plasma proteins and major spots of stained gels were identified by LC-MS/MS. Total RNA was isolated from testis, epididymis and vesicular glands and subjected to qPCR. At slaughter, scrotal circumference and testicular weight were 27.5 +/- 0.5cm and 109.5 +/- 6.0g, respectively. Seminiferous tubule (ST) diameter was 188.3 +/- 4.0m and each testis contained 1.9 +/- 0.1 Sertoli cells (x10(9)). Each Sertoli cell supported 0.1 +/- 0.01 A spermatogonia, 3.0 +/- 0.2 pachytene spermatocytes and 7.7 +/- 0.5 round spermatids/tubule cross section. Daily sperm production reached 5.6x10(6)cells/g of testis parenchyma. Testis size appeared as indicative of ST diameter and associated with epididymal measurements, as well as with the population of round spermatids and Sertoli cells/testis. Rams with heavier testes had greater daily sperm production and more Sertoli cells/testis. We detected 90.9 +/- 9.6 spots per 2-D gel of seminal plasma. Major seminal proteins were identified as ram seminal vesicle proteins at 14 and 22kDa, representing 16.2% and 12.8% of the total intensity of valid spots in the gels, respectively. Expression of both genes was greater in the vesicular glands as compared to testis and epididymis. Pixel intensity for those proteins in the 2-D gels was significantly correlated with seminal vesicle weight. This is the first description of the basic reproductive aspects of Morada Nova rams, including protein profiles of their seminal plasma. These findings will allow a better understanding of their reproductive physiology493409419CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESCeara State Agency for Technology and Scientific Development (FUNCAP

FERM domain interaction with myosin negatively regulates FAK in cardiomyocyte hypertrophy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Focal adhesion kinase (FAK) regulates cellular processes that affect several aspects of development and disease. The FAK N-terminal FERM (4.1 protein-ezrin-radixin-moesin homology) domain, a compact clover-leaf structure, binds partner proteins and mediates intramolecular regulatory interactions. Combined chemical cross-linking coupled to MS, small-angle X-ray scattering, computational docking and mutational analyses showed that the FAK FERM domain has a molecular cleft (similar to 998 angstrom(2)) that interacts with sarcomeric myosin, resulting in FAK inhibition. Accordingly, mutations in a unique short amino acid sequence of the FERM myosin cleft, FP-1, impaired the interaction with myosin and enhanced FAK activity in cardiomyocytes. An FP-1 decoy peptide selectively inhibited myosin interaction and increased FAK activity, promoting cardiomyocyte hypertrophy through activation of the AKT-mammalian target of rapamycin pathway. Our findings uncover an inhibitory interaction between the FAK FERM domain and sarcomeric myosin that presents potential opportunities to modulate the cardiac hypertrophic response through changes in FAK activity.81102110Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

FERM domain interaction with myosin negatively regulates FAK in cardiomyocyte hypertrophy

Focal adhesion kinase (FAK) regulates cellular processes that affect several aspects of development and disease. The FAK N-terminal FERM (4.1 protein-ezrin-radixin-moesin homology) domain, a compact clover-leaf structure, binds partner proteins and mediates intramolecular regulatory interactions. Combined chemical cross-linking coupled to MS, small-angle X-ray scattering, computational docking and mutational analyses showed that the FAK FERM domain has a molecular cleft (similar to 998 angstrom(2)) that interacts with sarcomeric myosin, resulting in FAK inhibition. Accordingly, mutations in a unique short amino acid sequence of the FERM myosin cleft, FP-1, impaired the interaction with myosin and enhanced FAK activity in cardiomyocytes. An FP-1 decoy peptide selectively inhibited myosin interaction and increased FAK activity, promoting cardiomyocyte hypertrophy through activation of the AKT-mammalian target of rapamycin pathway. Our findings uncover an inhibitory interaction between the FAK FERM domain and sarcomeric myosin that presents potential opportunities to modulate the cardiac hypertrophic response through changes in FAK activity.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2006/54878-3, 2007/55930-1, 2007/59442-1, 2008/53519-5, 2008/57805-2, 2010/02628-9]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Pesquisa (CNPq) [304366/2009-9, 475158/2010-5, 573672/2008-3, 559698/2009-7]Conselho Nacional de Pesquisa (CNPq