Mónica Carrera

Fast monitoring of species-specific peptide biomarkers using high-intensity-focused-ultrasound-assisted tryptic digestion and selected MS/MS ion monitoring.

A new strategy for the fast monitoring of peptide biomarkers is described. It is based on the use of accelerated in-solution trypsin digestions under an ultrasonic field provided by high-intensity focused ultrasound (HIFU) and the monitoring of several peptides by selected MS/MS ion monitoring in a linear ion trap mass spectrometer. The performance of the method was established for the unequivocal identification of all commercial fish species belonging to the Merlucciidae family. Using a particular combination of only 11 peptides, resulting from the HIFU-assisted tryptic digestion of the thermostable proteins parvalbumins, the workflow allowed the unequivocal identification of these closely related fish species in any seafood product, including processed and precooked products, in less than 2 h. The present strategy constitutes the fastest method for peptide biomarker monitoring. Its application for food quality control provides to the authorities an effective and rapid method of food authentication and traceability to guarantee the quality and safety to the consumers.

Extensive de novo sequencing of new parvalbumin isoforms using a novel combination of bottom-up proteomics, accurate molecular mass measurement by FTICR-MS, and selected MS/MS ion monitoring.

Parvalbumins (PRVB) (11.20-11.55 kDa) are considered the major fish allergens. In this work, we propose a novel strategy for extensive characterization of this group of proteins based on the integration of a classical Bottom-Up proteomics approach with accurate Mr determination by FTICR-MS of intact proteins and selected MS/MS ion monitoring (SMIM) of peptide mass gaps. For each PRVB, mass spectra obtained by LC-ESI-IT-MS/MS from two digests (trypsin, Glu-C) were de novo sequenced manually with help of two programs (PEAKS, DeNovoX). The deduced peptide sequences were arranged and the theoretical Mr for the resulting sequences was calculated. Experimental Mr for each PRVB was measured with high mass accuracy by FTICR-MS (0.05-4.47 ppm). The masses of several missing peptide gaps were estimated by comparing the theoretical and experimental Mr, and the MS/MS spectra corresponding to these ions were obtained by LC-ESI-IT-MS/MS in the SMIM scanning mode. Finally, all peptide sequences were combined to generate the final protein sequences. This approach allowed the complete de novo MS-sequencing of 25 new PRVB isoforms. These new sequences belong to 11 different species from the Merlucciidae family, organisms for which genomes remain unsequenced. This study constitutes the report accounting for the higher number of new proteins completely sequenced making use of MS-based techniques only.

Tackling proteome changes in the longissimus thoracis bovine muscle in response to pre-slaughter stress.

UNLABELLED Pre-slaughter stress has adverse effects on meat quality that can lead to the occurrence of Dark Firm Dry (DFD) meat in cattle. This study explores the previously uncharacterized proteome changes linked to pre-slaughter stress in the longissimus thoracis (LT) bovine muscle. Differential proteome profiles of DFD and normal (non-DFD) LT meat samples from male calves of the Rubia Gallega breed were assessed by 2-DE coupled to MS analysis (LC-MS/MS and MALDI TOF/TOF MS). A total of seven structural-contractile proteins (three different myosin light chain isoforms, two fast skeletal myosin light chain 2 isoforms, troponin C type 2 and cofilin-2) and three metabolism enzymes (triosephosphate isomerase, ATP synthase and beta-galactoside alpha-2,6-sialyltransferase) were found to have statistically significant differential abundance in sample groups. In addition, 2-DE in combination with the phosphoprotein-specific fluorescent dye Pro-Q DPS revealed that highly phosphorylated fast skeletal myosin regulatory light chain 2 isoforms underwent the most intense relative change in muscle conversion to DFD meat. Therefore, they appear to be the most sensitive biomarkers of stress just prior to slaughter in Rubia Gallega. Overall, these findings will facilitate a more integrative understanding of the biochemical processes associated with stress in cattle muscle and their effects in meat quality. BIOLOGICAL SIGNIFICANCE Pre-slaughter stress is a crucial factor in meat production. Animals destined for slaughter are stressed by a variety of endogenous and exogenous factors that negatively affect the complex post-mortem biochemical events underlying the conversion of muscle into meat. The study of the muscle proteome has a great relevance for understanding the molecular mechanisms associated with stress. However, there is no information available on the molecular changes linked to pre-slaughter stress in cattle on the proteome scale. Our study led to the identification of a number of candidate proteins associated with the response to pre-slaughter stress in the LT bovine muscle of the Rubia Gallega breed. The functions of those significantly changed proteins have a clear biological relationship with stress response. These findings contribute to a deeper insight into the molecular pathways that respond to stress in cattle.

Identification of the Major ACE-Inhibitory Peptides Produced by Enzymatic Hydrolysis of a Protein Concentrate from Cuttlefish Wastewater

The aim of this work was the purification and identification of the major angiotensin converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of a protein concentrate recovered from a cuttlefish industrial manufacturing effluent. This process consisted on the ultrafiltration of cuttlefish softening wastewater, with a 10 kDa cut-off membrane, followed by the hydrolysis with alcalase of the retained fraction. Alcalase produced ACE inhibitors reaching the highest activity (IC50 = 76.8 ± 15.2 μg mL−1) after 8 h of proteolysis. Sequential ultrafiltration of the 8 h hydrolysate with molecular weight cut-off (MWCO) membranes of 10 and 1 kDa resulted in the increased activity of each permeate, with a final IC50 value of 58.4 ± 4.6 μg mL−1. Permeate containing peptides lower than 1 kDa was separated by reversed-phase high performance liquid chromatography (RP-HPLC). Four fractions (A–D) with potent ACE inhibitory activity were isolated and their main peptides identified using high performance liquid chromatography coupled to an electrospray ion trap Fourier transform ion cyclotron resonance-mass spectrometer (HPLC-ESI-IT-FTICR) followed by comparison with databases and de novo sequencing. The amino acid sequences of the identified peptides contained at least one hydrophobic and/or a proline together with positively charged residues in at least one of the three C-terminal positions. The IC50 values of the fractions ranged from 1.92 to 8.83 μg mL−1, however this study fails to identify which of these peptides are ultimately responsible for the potent antihypertensive activity of these fractions.

Protein biomarker discovery and fast monitoring for the identification and detection of Anisakids by parallel reaction monitoring (PRM) mass spectrometry

UNLABELLED Anisakids are fish-borne parasites that are responsible for a large number of human infections and allergic reactions around the world. World health organizations and food safety authorities aim to control and prevent this emerging health problem. In the present work, a new method for the fast monitoring of these parasites is described. The strategy is divided in three steps: (i) purification of thermostable proteins from fish-borne parasites (Anisakids), (ii) in-solution HIFU trypsin digestion and (iii) monitoring of several peptide markers by parallel reaction monitoring (PRM) mass spectrometry. This methodology allows the fast detection of Anisakids in <2h. An affordable assay utilizing this methodology will facilitate testing for regulatory and safety applications. SIGNIFICANCE The work describes for the first time, the Protein Biomarker Discovery and the Fast Monitoring for the identification and detection of Anisakids in fishery products. The strategy is based on the purification of thermostable proteins, the use of accelerated in-solution trypsin digestions under an ultrasonic field provided by High-Intensity Focused Ultrasound (HIFU) and the monitoring of several peptide biomarkers by Parallel Reaction Monitoring (PRM) Mass Spectrometry in a linear ion trap mass spectrometer. The workflow allows the unequivocal detection of Anisakids, in <2h. The present strategy constitutes the fastest method for Anisakids detection, whose application in the food quality control area, could provide to the authorities an effective and rapid method to guarantee the safety to the consumers.

Determination of the geographical origin of all commercial hake species by Stable Isotope Ratio (SIR) analysis

The determination of the geographical origin of food products is relevant to comply with the legal regulations of traceability, to avoid food fraud, and to guarantee food quality and safety to the consumers. For these reasons, stable isotope ratio (SIR) analysis using an isotope ratio mass spectrometry (IRMS) instrument is one of the most useful techniques for evaluating food traceability and authenticity. The present study was aimed to determine, for the first time, the geographical origin for all commercial fish species belonging to the Merlucciidae family using SIR analysis of carbon (δ13C) and nitrogen (δ15N). The specific results enabled their clear classification according to the FAO (Food and Agriculture Organization of the United Nations) fishing areas, latitude, and geographical origin in the following six different clusters: European, North African, South African, North American, South American, and Australian hake species.

Discrimination of South African Commercial Fish Species (Merluccius capensis and Merluccius paradoxus) by LC-MS/MS Analysis of the Protein Aldolase

Analysis of the protein aldolase using proteomic methodologies allowed the discrimination of the Cape hakes, Merluccius capensis and Merluccius paradoxus. A classical bottom-up proteomics approach, consisting of two-dimensional gel electrophoresis, tryptic in-gel digestion, and LC-MS/MS analyses, was performed for the characterization and de novo sequencing of the aldolase spots. The sequences discovered presented a high degree of homology with the aldolase A sequence from the teleostei Tetraodon nigroviridis. De novo sequencing of several MS/MS spectra from the aldolase A spots allowed the characterization of two species-specific peptide biomarkers. One of them was unique to Merluccius capensis while the other was present only in Merluccius paradoxus. These peptide biomarkers can be used to discriminate between both species of Cape hakes.

Fast Global Phosphoproteome Profiling of Jurkat T Cells by HIFU-TiO2-SCX-LC-MS/MS

We propose a new workflow for fast phosphoproteome profiling. The workflow is based on the use of accelerated in-solution trypsin digestion under an ultrasonic field provided by high-intensity focused ultrasound (HIFU) combined with an inverse strategy based on TiO2 selective phosphopeptide enrichment, fractionation by strong cation exchange chromatography (SCX) and analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) using a high-resolution mass spectrometer. The performance of the method was established for the global phosphoproteome analysis of unstimulated human Jurkat leukemia T cells (E6.1). Using this accelerated workflow, 15367 phosphorylation sites from 13029 different phosphopeptides belonging to 3163 different phosphoproteins were efficiently identified with high-throughput and reproducibility in less than 15 h. The functional analysis revealed significant phosphorylation-based networks that are implicated in immune function and tumor development pathways. The present strategy, HIFU-TiO2-SCX-LC-MS/MS, is the fastest analytical method reported to date for generating large-scale phosphoproteomics data sets (<15 h).

Novel Peptide Biomarker Discovery for Detection and Identification of Bacterial Pathogens by LC-ESI-MS/MS

9 paginas, 1 tabla, 7 figuras.-- This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Quantification of proteome changes in bovine muscle from two-dimensional electrophoresis data

Proteome changes in the longissimus thoracis bovine muscle in response to pre-slaughter stress were assessed on the basis of two-dimensional electrophoresis (2-DE) data. In this study, the bootstrap resampling statistical technique and a new measure of relative change of the volume of 2-DE protein spots are shown to be more efficient than commonly used statistics to reliably quantify changes in protein abundance in stress response. The data are supplied in this article and are related to “Tackling proteome changes in the longissimus thoracis bovine muscle in response to pre-slaughter stress” by Franco et al. [1].