Quantification in Proteomics. 5th Short Mass Spectrometry Courses 1/4/2016 Jana Březinová Mass Spectrometry IOCB AS CR - PDF

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Quantification in Proteomics 5th Short Mass Spectrometry Courses 1/4/2016 Jana Březinová Mass Spectrometry IOCB AS CR Presentation Outline Bottom-up approach in proteomics Quantification methods Labelling

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Quantification in Proteomics 5th Short Mass Spectrometry Courses 1/4/2016 Jana Březinová Mass Spectrometry IOCB AS CR Presentation Outline Bottom-up approach in proteomics Quantification methods Labelling techniques Metabolic labelling SILAC Chemical labelling dimethyl labelling Label-free techniques MS approach MaxQuant LFQ MS/MS all approach SWATH Targeted approach SRM How to design your own experiment? Quantification in Proteomics Bottom-up approach in proteomics Quantification methods Labelling techniques Metabolic labelling SILAC Chemical labelling dimethyl labelling Label-free techniques MS approach MaxQuant LFQ MS/MS all approach SWATH Targeted approach SRM How to design your own experiment? Bottom-up proteomics https://upload.wikimedia.org/wikipedia/commo ns/1/1f/mass_spectrometry_protocol.png Limitations of Bottom-up Approach Protein level information is inherently lost Proteins are quantified indirectly, their ratios are inferred from peptides after digestion. Protein isoforms impose a problem to protein quantitation. Close inspection of data on peptide level is required. Quantification in Proteomics Bottom-up approach in proteomics Quantification methods Labelling techniques Metabolic labelling SILAC Chemical labelling dimethyl labelling Label-free techniques MS approach MaxQuant LFQ MS/MS all approach SWATH Targeted approach SRM How to design your own experiment? MS quantification Based on peak heights or areas MS level quantification: XIC of precursor peptide(s) m/z MS/MS level quantification: XIC Of fragment peptide ion(s) m/z TIC MS level XIC of precursor Fragmentation spectrum MS/MS level XIC of fragments MS quantification in proteomics Often only relative determination of quantity Labelling techniques A mass tag (label) is introduced into the protein or peptide. Compared samples are mixed together and analyzed. The introduced mass shift enables relative quantitation. Label-free techniques The mass of the protein or peptide remains unchanged, samples are analysed separately. https://www.thermofisher.com/cz/en/home/lifescience/protein-biology/protein-biology-learningcenter/protein-biology-resource-library/pierce-proteinmethods/quantitative-proteomics.html Quantification in Proteomics Bottom-up approach in proteomics Quantification methods Labelling techniques Metabolic labelling SILAC Chemical labelling dimethyl labelling Label-free techniques MS approach MaxQuant LFQ MS/MS all approach SWATH Targeted approach SRM How to design your own experiment? Labelling techniques Stable isotopes Differential mass labels Heavy: 13 C, 15 N, 18 O, 2 H Light: 12 C, 14 N, 16 O, 1 H Introduction of single elements Trypsin digestion in H 2 18 O 15 N labelling of cell cultures Introduction of compounds labelled by multiple heavy isotopes Stable isotope labelling of amino acids in cell culture (SILAC) Isobaric tag for relative and absolute quantitation (itraq) Presumptions Equal behavior under chromatographic conditions corresponding H/L labeled peptides elute at the same time Equal MS sampling probability of the isotopes during their elution window Labelling techniques Metabolical Eg. SILAC Chemical Eg. Dimethyl labelling Quantification in Proteomics Bottom-up approach in proteomics Quantification methods Labelling techniques Metabolic labelling SILAC Chemical labelling dimethyl labelling Label-free techniques MS approach MaxQuant LFQ MS/MS all approach SWATH Targeted approach SRM How to design your own experiment? Metabolic Labelling SILAC Stable isotope labeling by amino acids in cell culture Cell culture is grown on a medium containing either only heavy or light AAs (Arg, Lys) auxotrophy required Labelled AAs are used as protein building blocks After at least 5 cell cycles a nearly full incorporation of the heavy AAs is achieved Label incorporation needs to be monitored Eliminated influence of sample preparation variations on quantification https://www.broadinstitute.org/scientific-community/science/platforms/proteomics/silac SILAC Incorporation LC-MS/MS quantitation result of a selected protein from a heavy labeled cell culture First step: Incorporation level of heavy AAs ( 13 C 15 N Arg, Lys) into the cell culture proteins Protein selection Peptide selection H/L ratio = 35 i.e. incorporation level 97 % SILAC Advantages Samples are mixed early in experiment accounts for any sample losses Suited also when extensive sample prep is required Both shotgun and targeted approach Disadvantages Auxotrophy for Lys, Arg Easily applicable only to cell cultures Metabolic conversion of Arg to Pro Limited multiplicity Expensive Quantification in Proteomics Bottom-up approach in proteomics Quantification methods Labelling techniques Metabolic labelling SILAC Chemical labelling dimethyl labelling Label-free techniques MS approach MaxQuant LFQ MS/MS all approach SWATH Targeted approach SRM How to design your own experiment? Chemical labelling Cysteine labelling techniques ICAT Isotope coded affinity tag Primary amine labelling techniques Dimethyl labelling itraq (AB Sciex) TMT (Thermo) Dimethyl labelling Reaction of N-termini and ε-amino group of lysine with formaldehyde followed by reduction with sodium cyanoborohydride Boersema, P. J., et al. Triplex protein quantification based on stable isotope labeling by peptide dimethylation applied to cell and tissue lysates. Proteomics. 8, 2008, pp Dimethyl labelling Advantages Cheap and easily accessible reagents Reaction Fast In solution after digestion Disadvantages Other primary amines may react with formaldehyde avoid Tris, Am. Bic, use TEAB All steps prior mixing of samples may influence your results optimisation required Quantification in Proteomics Bottom-up approach in proteomics Quantification methods Labelling techniques Metabolic labelling SILAC Chemical labelling dimethyl labelling Label-free techniques MS approach MaxQuant LFQ MS/MS all approach SWATH Targeted approach SRM How to design your own experiment? Label-free techniques Less expensive (no labels) Practical for large animal or biomarker studies Unrestricted number of compared samples Sample prepation needs to be highly reproducible internal standards or house-keeping proteins MaxQuant Label-free Quantitation (MaxLFQ) Quantification on MS-level Unidentified peptides in one sample are matched to expectant m/z and retention time from another more intensive one Advanced normalization also for fractionated samples Standard data-dependent acquisition MS-level quantification Advantages Precursor ion intensity Fragment ion intensity Disadvantages Instrument with high mass resolution is essential Less selective than MS/MS based quantification methods Quantification in Proteomics Bottom-up approach in proteomics Quantification methods Labelling techniques Metabolic labelling SILAC Chemical labelling dimethyl labelling Label-free techniques MS approach MaxQuant LFQ MS/MS all approach SWATH Targeted approach SRM How to design your own experiment? MS/MS-level quantitation - SWATH acquisition TripleTOF 5600 Precursor selection window (in DDA single m/z) Fragmentation in the collision cell MS/MS scan of fragments originating from all precursors from the selection window SWATH MS/MS ALL Acquisition SWATH Advantages Enables quantititation of previously not considered proteins Simplifies SRM method development choice of precursor ions is less elaborate Disadvantages Large and complex data files Spectral library needs to be generated in a separate acquisition run Quantification in Proteomics Bottom-up approach in proteomics Quantification methods Labelling techniques Metabolic labelling SILAC Chemical labelling dimethyl labelling Label-free techniques MS approach MaxQuant LFQ MS/MS all approach SWATH Targeted approach SRM How to design your own experiment? Targeted analysis Precursor selection (Peptide m/z) Q1 Fragmentation in the collision cell Q2 Fragment ion scan (Peptide fragment m/z) Q3 Quantitation SRM principle Chromatogram choice of peptide retention time MS spectrum m/z of precursor ion (Q1) MS/MS fragmentation spectrum (Q3) Quantification in Proteomics Bottom-up approach in proteomics Quantification methods Labelling techniques Metabolic labelling SILAC Chemical labelling dimethyl labelling Label-free techniques MS approach MaxQuant LFQ MS/MS all approach SWATH Targeted approach SRM How to design your own experiment? How to design your own experiment? What do you want to quantify? TARGETED vs. DISCOVERY analysis Quantification of one protein you as many as possible? What do you know about the proteins and the sample? Do you expect any modifications? Do you need enrichment? How precise and accurate need to be the results? LABEL vs. LABEL-FREE techniques What is the biological variation you expect? Do you expect major changes in protein levels in your experiment? How many replicates are you able to perform? Can you validate the results using a complementary method? Try to identify the critical steps in your sample preparation workflow How to design your own quant. experiment? Sample characterization Type Origin Amount Proteins Digestion In-solution Cell Culture Bacterial 10ug Cytosolic Deoxycholate RapiGest SF Tissue Mammalian 10ug Membrane Filter-aided (efasp) SDS+deoxycholic acid Purified protein Pitfalls in protein quantification Accuracy of quantitation is limited by Protein Isoforms: part of their protein sequence is shared but belongs to multiple proteins Unwanted modifications occuring during sample preparation (oxidation of Met, incomplete labeling etc.) Deuterium labelled peptides may behave differently in reversed-phase chromatography Reproducibility of quantitation is limited by Variations in experimental conditions, sample preparation, sample complexity DDA: Data dependent acquisition interferences influence the m/z picking for MS/MS fragmentation Try to identify the critical steps in your sample preparation workflow Thank you for your attention Do not hesitate to contact us Martin Hubálek, Ph.D. Jana Březinová Karel Rücker Lab assistant
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