Material que habrá de ser revisado para esta unidad:
Libros:
1. Daniel C. Liebler. Introduction to Proteomics. Tools for the New Biology
2002.
- Cap. 5. Protein Digestion Techniques
- Cap. 7. Protein Identification by Peptide Mass Fingerprinting
- Cap. 4. Amino Acids: The building bloks of protein
3. M.R. Wilkins, R.D. Appel, K.L. Wilkins and D.F. Hochstrasser (Eds). Proteome Research: Concepts, Technology and Application. Springer-Verlag Berlin Heidelberg. 2007.
- Cap. 3. Protein identification in proteomics. P. Hernández, P Binz, M.R. Wilkins.
- Bischoff R, Schlüter H. Aminoacids: Chemistry, funcionality and selected non-enzymatic post-translational modifications.J Proteomics. 2012 Apr 18;75(8):2275-96. http://www.sciencedirect.com/science/article/pii/S1874391912000838
- The ultimate goal of proteomics is determination of the exact chemical composition of protein species, including their complete amino acid sequence and the identification of each modified side chain, in every protein in a biological sample and their quantification.We are still far from achieving this goal due to limitations in analytical methodology and data analysis but also due to the fact that we surely have not discovered all amino acid modifications that occur in nature. To detect modified side chains and to discover new, still unknown amino acid derivatives, an understanding of the chemistry of the reactive groups of amino acids is mandatory. This tutorial focuses on the chemistry of the aminoacid side chains and addresses non-enzymatic modifications. By highlighting some exemplary reactions a glimpse of the huge diversity of modified amino acids provides the reader with sufficient insight into amino acid chemistry to raise the awareness for unexpected side chain modifications. We further introduce the reader to a terminology, which enables the comprehensive description of the exact chemical composition of a protein species, including its full amino acid sequence and all modifications of its amino acid side chains. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP number 10).
Biron DG, Brun C, Lefevre T, Lebarbenchon C, Loxdale HD, Chevenet F, Brizard JP, Thomas F The pitfalls of proteomics experiments without the correct useof bioinformatics tools. Proteomics. 2006 Oct;6(20):5577-96.- http://onlinelibrary.wiley.com/doi/10.1002/pmic.200600223/abstract
- The elucidation of the entire genomic sequence of various organisms, from viruses to complex metazoans, most recently man, is undoubtedly the greatest triumph of molecular biology since the discovery of the DNA double helix. Over the past two decades, the focus of molecular biology has gradually moved from genomes to proteomes, the intention being to discover the functions of the genes themselves. The postgenomic era stimulated the development of new techniques (e.g. 2-DE and MS) and bioinformatics tools to identify the functions, reactions, interactions and location of the gene products in tissues and/or cells of living organisms. Both 2-DE and MS have been very successfullyemployed to identify proteins involved in biological phenomena(e.g.immunity,cancer,host–parasite interactions, etc.), although recently, several papers have emphasised the pitfalls of 2-DE experiments, especially in relation to experimental design, poor statistical treatment and the high rate of ‘false positive’ results with regard to protein identification. In the light of these perceived problems, we review the advantages and misuses of bioinformatics tools – from realisation of 2-DE gels to the identification of candidate protein spots – and suggest some useful avenues to improve the quality of 2-DE experiments. In addition, we present key steps which, in our view, need to be to taken into consideration during such analyses. Lastly, we present novel biological entities named ‘interactomes’, and the bioinformatics tools developed to analyse the large protein–protein interaction networks they form, along with several new perspectives of the field.
Encarnación S, Hernández M, Martínez-Batallar G, Contreras S, Vargas Mdel C,Mora J. Comparative proteomics using 2-D gel electrophoresis and massspectrometry as tools to dissect stimulons and regulons in bacteria withsequenced or partially sequenced genomes. Biol Proced Online. 2005;7:117-35.http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1190382/We propose two-dimensional gel electrophoresis (2-DE) and mass spectrometry to define the protein components of regulons and stimulons in bacteria, including those organisms where genome sequencing is still in progress. The basic 2-DE protocol allows high resolution and reproducibility and enables the direct comparison of hundreds or even thousands of proteins simultaneously. To identify proteins that comprise stimulons and regulons, peptide mass fingerprint (PMF) with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS) analysis is the first option and, if results from this tool are insufficient, complementary data obtained with electrospray ionization tandem-MS (ESI-MS/MS) may permit successful protein identification. ESI-MS/MS and MALDI-TOF-MS provide complementary data sets, and so a more comprehensive coverage of a proteome can be obtained using both techniques with the same sample, especially when few sequenced proteins of a particular organism exist or genome sequencing is still in progress.
Steen H,Mann M. The ABC's (and XYZ's)of peptide sequencing.Nat Rev Mol Cell Biol.2004 Sep;5(9):699-711. http://www.nature.com/nrm/journal/v5/n9/full/nrm1468.html
Proteomics is an increasingly powerful and indispensable technology in molecular cell biology. It can be used to identify the components of small protein complexes and large organelles, to determine post-translational modifications and in sophisticated functional screens.The key — but little understood — technology in mass-spectrometry-based proteomics is peptide sequencing, which we describe and review here in an easily accessible format.Article Source: Modular Mass Spectrometric Tool for Analysis of Composition and Phosphorylation of Protein Complexes(2007) Modular Mass Spectrometric Tool for Analysis of Composition and Phosphorylation of Protein Complexes. PLoS ONE 2(4): e358. doi:10.1371/journal.pone.0000358The combination of high accuracy, sensitivity and speed of single and multiple-stage mass spectrometric analyses enables the collection of comprehensive sets of data containing detailed information about complex biological samples. To achieve these properties, we combined two high-performance matrix-assisted laser desorption ionization mass analyzers in one modular mass spectrometric tool, and applied this tool for dissecting the composition and post-translational modifications of protein complexes. As an example of this approach, we here present studies of the Saccharomyces cerevisiae anaphase-promoting complexes (APC) and elucidation of phosphorylation sites on its components. In general, the modular concept we describe could be useful for assembling mass spectrometers operating with both matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) ion sources into powerful mass spectrometric tools for the comprehensive analysis of complex biological samples.Thelen JJ, Miernyk JA. The proteomic future: where mass spectrometry should be taking us. Biochem J. 2012 Jun 1;444(2):169-81.http://www.biochemj.org/bj/444/0169/bj4440169.htmA newcomer to the -omics era, proteomics, is a broad instrument-intensive research area that has advanced rapidly since its inception less than 20 years ago. Although the ‘wet-bench’ aspects of proteomics have undergone a renaissance with the improvement in protein and peptide separation techniques, including various improvements in two-dimensional gel electrophoresis and gel-free or off-gel protein focusing, it has been the seminal advances in MS that have led to the ascension of this field. Recent improvements in sensitivity, mass accuracy and fragmentation have led to achievements previously only dreamed of, including whole-proteome identification, and quantification and extensive mapping of specific PTMs (post-translational modifications). With such capabilities at present, one might conclude that proteomics has already reached its zenith; however, ‘capability’ indicates that the envisioned goals have not yet been achieved. In the present review we focus on what we perceive as the areas requiring more attention to achieve the improvements in workflow and instrumentation that will bridge the gap between capability and achievement for at least most proteomes and PTMs. Additionally, it is essential that we extend our ability to understand protein structures, interactions and localizations. Towards these ends, we briefly focus on selected methods and research areas where we anticipate the next wave of proteomic advances.Römpp A, Spengler B. Mass spectrometry imaging with high resolution in mass
and space. Histochem Cell Biol. 2013 Jun;139(6):759-83.
http://link.springer.com/article/10.1007%2Fs00418-013-1097-6
Mass spectrometry (MS) imaging links molecular information and the spatial distribution of analytes within a sample. In contrast to most histochemical techniques, mass spectrometry imaging can differentiate molecular modifications and does not require labeling of targeted compounds. We have recently introduced the first mass spectrometry imaging method that provides highly specific molecular information (high resolution and accuracy in mass) at cellular dimensions (high resolution in space). This method is based on a matrix-assisted laser desorption/ionization (MALDI) imaging source working at atmospheric pressure which is coupled to an orbital trapping mass spectrometer. Here, we present a number of application examples and demonstrate the benefit of ‘mass spectrometry imaging with high resolution in mass and space.’ Phospholipids, peptides and drug compounds were imaged in a number of tissue samples at a spatial resolution of 5–10 μm. Proteins were analyzed after on-tissue tryptic digestion at 50-μm resolution. Additional applications include the analysis of single cells and of human lung carcinoma tissue as well as the first MALDI imaging measurement of tissue at 3 μm pixel size. MS image analysis for all these experiments showed excellent correlation with histological staining evaluation. The high mass resolution (R = 30,000) and mass accuracy (typically 1 ppm) proved to be essential for specific image generation and reliable identification of analytes in tissue samples. The ability to combine the required high-quality mass analysis with spatial resolution in the range of single cells is a unique feature of our method. With that, it has the potential to supplement classical histochemical protocols and to provide new insights about molecular processes on the cellular level.
- Un aminoácido (aa) es un compuesto nitrogenado formado por un grupo amino (-NH2) y un grupo carboxilo (-COOH) unidos a un mismo átomo de carbono (C-α), en este C- α se encuentra unida una cadena lateral (R). (Figura1)
Los diferentes aa poseen ciertas características
químicas como: carácter básico o ácido, hidrofilicidad o hidrofobicidad; que están
dictadas por la naturaleza de las cadenas laterales que los constituyen y que van a influir en la estructura de una proteína y por ende
en su función. Los grupos laterales pueden ser no polares
(sin diferencia de carga entre distintas zonas del grupo), polares pero con
cargas balanceadas de modo tal que el grupo lateral en conjunto es neutro, o
cargados, negativa o positivamente. (Tabla 1)
En términos generales, los aa son considerados anfóteros ya que pueden actuar como ácidos o bases dependiendo del pH del medio. El carácter ácido/básico de los aminoácidos hacen que se presente una carga negativa (-COO-) y una positiva(-NH3+ ) por lo cual son denominados zwitteriones; en esta condición la carga neta del aa es cero(pI: punto isoeléctrico). El pI de cada aa depende del pKa delos grupos químicos de la cadena lateral y del pH del medio en el que se encuentra. El índice de hidropatía* de un aa es considerado como la medida de la energía (KJ/mol) que se debe usar para transferirlo de un medio hidrófobo(valores negativos) a un medio hidrofilico (valores positivos), así por ejemplo, una proteína de membrana estará constituida por aa hidrófobos en la parte embebida en la membrana y por aa hidrofílicos al exterior de ella (citosol e interior de la célula). (Tabla 2).
Es importante tener en consideración dichas características ya que en ellas se basan las técnicas de purificación y separación de proteínas, por ejemplo: la cromatografía en sus diferentes modalidades (intercambio iónico, afinidad exclusión molecular) y la electroforesis en una y dos dimensiones (separación por masa y carga).
La masa
isotópica de los aa es la masa
considerada con el isotopo C12, y la cual es considerada en la
identificación por espectrometría de masas (MS). Por medio de la MS se han
logrado identificar diferentes modificaciones en los aa, ya que hay cambios en la masa y/o carga que presentan los aa modificados en
comparación con la masa isotópica del mismo aa sin modificar.
La reacción más frecuente que sufren los
aa es la formación del enlace peptídico durante el proceso de traducción de las proteínas. En los polipéptidos los aa se enlazan entre el α –amino (-NH2)
de un aminoácido y el
α –carboxilo (-COOH)
de un segundo aminoácido, este enlace es de tipo covalente (Figura 2).
Estas reacciones se repiten muchas veces hasta integrar una proteína. Las modificaciones postraduccionales (MPT), el procesamiento proteolítico y el splicing de proteínas resultan en un constante cambio en el proteoma, que es mucho mas dinámico y complejo que el genoma correspondiente.
Cuando existe una modificación en una
proteína; diferente a las habituales, puede cambiar aparatosamente la función
biológica de esta. Las
modificaciones en las cadenas laterales dependen de la reactividad de los
grupos químicos , en especial del grado de electronegatividad que tengan (ya que los puede hacer más
electrófilos o neutrófilos), de la exposición y suceptibilidad para reaccionar
y del ambiente alrededor del aa. Cabe mencionar que en algunos casos hay
modificaciones en las que se adicionan aa completos a una proteína que pueden
conferirle alguna función en específico; estos residuos se añaden al extremo
C-terminal y pueden o no ser reversibles
(por ejemplo, la arginilización y glutaminación en proteínas son irreversibles).
En las cadenas laterales de Lys, His, Cys, Asp,
Glu y Tyr los grupos funcionales son potentes nucleófilos que in vivo van a
ser atacados por electrófilos presentes en diferentes co-sustratos presentes en el ambiente celular
como: ATP, Acetil Co-A, NAD+, etc…, causantes de las modificaciones que se dan
naturalmente.
El orden relativo de nucleofilicidad de los grupos funcionales de los aa es:
R-S->R-NH2>R-COO-=R-O-
. Donde hay que destacar que los grupos aril y
alquilo son grupos con menor capacidad de reacción.
Tomando en cuenta la nucleofílicidad de los
distintos grupos funcionales se puede ordenar a los aa por su capacidad para
reaccionar en forma decreciente: Cys, Met,
Lys, Arg, Gln, Asn, His, Glu, Asp, Ser, Thr, Tyr, Trp, Pro y Gly.
La Cys y Met sufren reacciones de oxidación mas frecuentemente debido a l grupo sulfuro
que poseen en su cadena lateral. En la
célula las reacciones en las que más comúnmente intervienen los tioles son en
las que se encargan de detoxificación por xenóbioticos. Las principales
modificaciones de los aminoácidos más reactivos se observan en la Tabla 3.
Puede observarse que el numero de modificaciones que puede sufrir un aa es variable, esto puede explicarse en función de la composición de la cadena lateral donde esta localizado el grupo funcional, lo que puede provocar una mayor o menor susceptibilidad a ciertas modificaciones. Actualmente existen anticuerpos monoclonales que pueden identificar modificaciones, estos pueden identificar una modificación en cierta posición de una secuencia en diferentes organismos, haciendo más fácil la identificación de una proteína modificada (p.ej. fosforilada).
Además, los aa en una proteína no solo
son blancos de las modificaciones que se pueden dar postraduccionalmente dentro
de la célula, sino que pueden
modificarse químicamente (derivatización) por medio de reactivos durante el
tratamiento para identificar una proteína por MS, estas modificaciones pueden
servir para seleccionar y enriquecer
péptidos que son de interés.
Por todo lo anterior se concluye que es
de vital importancia conocer las posibles modificaciones que pueden sufrir los
aa para lograr un correcto análisis e identificar las proteínas
y en su caso a los aa que se están estudiando.
Bibliografía.
Rainer
Bischoff, Hartmut Schuluter. ““Amino acids: Chemistry, functionality and
selected non-enzymatic post-translational modifications”, Journal of
Proteomics, 75:2275-2296 (2012).
*Werner Muller-Esterl.”Bioquimica
Fundamentos para la medicina y Ciencias
de la vida”, Editorial Reverte Pág.352 (2008).
.jpg)
