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Introduction to Protein Chemistry. Gustavo de Souza IMM, OUS. October 2013. Relevance of the Proteome. Relevance of the Proteome. «The recipe of life». X. Chocolate cake : Egg Flour Sugar Baker’s yeast Chocolate. Biological relevance lies on how genes
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Introduction to Protein Chemistry Gustavo de SouzaIMM, OUS October 2013
Relevance of the Proteome «The recipe of life» X • Chocolatecake: • Egg • Flour • Sugar • Baker’syeast • Chocolate Biological relevance lies on how genes are expressed and translated to proteins, not if genes are present or not
Primary Structure >sp|F2Z333|CA233_HUMAN Fibronectin type-III domain-containing transmembrane protein C1orf233 MRAPPLLLLLAACAPPPCAAAAPTPPGWEPTPDAPWCPYKVLPEGPEAGGGRLCFRSPAR GFRCQAPGCVLHAPAGRSLRASVLRNRSVLLQWRLAPAAARRVRAFALNCSWRGAYTRFP CERVLLGASCRDYLLPDVHDSVLYRLCLQPLPLRAGPAAAAPETPEPAECVEFTAEPAGM QDIVVAMTAVGGSICVMLVVICLLVAYITENLMRPALARPGLRRHP
Primary Structure - Folding >sp|F2Z333|CA233_HUMAN Fibronectin type-III domain-containing transmembrane protein C1orf233 MRAPPLLLLLAACAPPPCAAAAPTPPGWEPTPDAPWCPYKVLPEGPEAGGGRLCFRSPAR GFRCQAPGCVLHAPAGRSLRASVLRNRSVLLQWRLAPAAARRVRAFALNCSWRGAYTRFP CERVLLGASCRDYLLPDVHDSVLYRLCLQPLPLRAGPAAAAPETPEPAECVEFTAEPAGM QDIVVAMTAVGGSICVMLVVICLLVAYITENLMRPALARPGLRRHP
Folding Proteins can adopt only a limited number of different protein folds
What is a «protein sample» in proteomics? RNA-binding protein modules
Take home message • Proteins are the functionally active molecule in a cell. • They possess a high degree of chemical and structural heterogeneity. • 3. Heterogeneity interfere in how a protein sample can be analyzed
Challenges in Protein and Proteomic Analysis Gustavo de SouzaIMM, OUS October 2013
A dangerous idea… One gene, one protein Homo sapiens
A less dangerous idea One gene, some proteins (let’s say average 5 per gene) Homo sapiens
Complexity of Protein Samples in Eukaryotes PTMs (modifications thatcontrol conformationchanges in histones)
An even less dangerous idea One protein, possible 8 modification sites Homo sapiens
But in reality… • One specific cell does NOT express all genes at once! • Several transcriptomics studies indicated that the cells under study have ~14000 transcripts at a certain time Homo sapiens
Proteome Dynamics B C A Genome is a relatively static element of an organism, the proteome is changing accordingly to cell type, cell stage developmet, response to stress, etc.
Proteome dynamics within the same cell Proteome can change with the least of the stimuli within a cell
Proteome chemical heterogeneity DNA - Negatively charged molecule Has the same phisico-chemical featuresregardless of: its nucleotide sequence,its tissue source, its donor source, thespecies of the donor, etc.
Proteome chemical heterogeneity Membrane proteins
Proteome dynamic range Genome Mostly, individual genes are observed equimolar amounts in a DNA molecule Transcription/Translation Protein concentration within a cellis unique to each individual protein Difference between most and leastabundant molecule = dynamic range
Proteome dynamic range Dynamic range of a proteome estimated to be around 10e8 (in serum isbelieved to be over 10e10) Geiger et al., MCP 2012
Proteome dynamic range Difference between the most and lowest abundant proteins Cytoskeleton (Actin, tubulin, vimentin) Protein abundance Chaperons (hsp60, hsp70, calreticulin) Metabolism (glycolisis, ribosomal) Mytochondria (respiratory chain) Structure Nucleus (histones) Organelles Signalling pathway proteins, transcription factors, etc Protein GO classification
Instrumentation Aebersold & Mann, Nature 2003
Instrumentation • Instrumentations with different hardware generate different types of raw data. • Different brands developed different computer formats, with need for different libraries to read the file. • Which lead to development of a whole bunch of specific software usingspecific computational protocols. • Lack of standard routine.
Take home message • Proteomic composition is at least 6x more complex than the genomic composition of a cell, if only number of entities is considered. • It is an ever changing feature, limited by spatial and time constrains. • Chemical properties and dynamic range has an relevant impact in success rate of identification using proteomic methods. • Instrumentation and Analysis is not standardized.
Introduction to Mass SpectrometryInterpreting peptide/protein data Gustavo de SouzaIMM, OUS October 2013
Lets talk about…physics 3D Quadrupole ion trap Linear Quadrupole ion trap
What is it? • Instrument which can detect the mass-to-charge (m/z)of ions (or ionized molecules). • Ionization must generate ions in gas-phase • Ion detection is proportional to its abundance in the sample • MS performs at extremely low pressures (vacuum) • - Any molecule is ionizable: small organic/inorganic chemicals(less than 300 Da), average sized peptides or DNAfragments, intact proteins.
Mass Spectrometry Scheme Ion Source Mass Analyzer Inlet Detector LC MALDI ES Time-of-Flight Quadrupole Ion Trap
Isotopes Normally observed in nature. Mass difference = 1 Da
What to expect from a mass spectrum Intensity m/z Avogadro number = 6.022x10e23 /mol
Peptide mass spectrum • Isotopes (12C, 13C, 14N, 15N)