Principles of Proteomic Methods

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1 Principles of Proteomic Methods Marc Wilkins School of Biotechnology and Biomolecular Sciences

2 What is Proteomics? - proteomics is the study of proteomes - proteomics aims to: separate, identify and characterise proteins on a large scale localise proteins in organelles, cells, tissues define levels of proteins in cells / tissues & how these change investigate protein interactions elucidate protein function, pathways and interrelationships protein separation and quantitation protein identification & characterisation protein pathways and networks

3 Separation of Proteins in a Proteome Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) described by O Farrell and Klose, 1975 first separates proteins by charge then separates proteins by size (mass) is the highest resolution separation technique for proteins. Why?

4 The Big 2-D Gel 10,345 protein spots in testis Klose, J., Kobalz, U., Electrophoresis 1995, 16,

proteins that are present or absent - changes in protein expression levels These changes may

5 Finding Changes in the Proteome by 2-D Gels N N D D D N Samples of normal (N) and diseased (D) 2-D gels of samples changes in protein spots are revealed Comparison of 2-D gels can reveal: - proteins that are present or absent - changes in protein expression levels These changes may be indicative of a disease or phenotype. But. difficult to do well. And what are the proteins?

6 Protein Identification by Mass Spectrometry What is mass spectrometry? a technique that measures the mass of molecules Remember the mass of carbon = 12 Daltons the mass of nitrogen = 14 the mass of oxygen = 16 The mass of glycine (an amino acid)? C 2 O 2 NH 5 = 79 Daltons The mass of this protein? MVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMG NPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCV LAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH 15,998 Daltons

7 Peptide Mass Fingerprinting a one at a time method for protein identification TYGGAAR EHICLLGR GPGFK GANR PSTTGVEMFR protein from 2-D gel Digestion of protein to peptides mixture of peptides match against theoretically digested proteins in database mass spectrometry to measure peptide masses

Tandem Mass Spectrometry Protein Identification - Separate protein to purity - Cut protein to peptides - Separate peptides by chromatography - Ionise in mass spectrometer - Measure

8 Tandem Mass Spectrometry Protein Identification - Separate protein to purity - Cut protein to peptides - Separate peptides by chromatography - Ionise in mass spectrometer - Measure peptide masses - Select peptides and fragment - Repeat for all peptides - Identify protein by database matching peptide mass and fragment masses Pandey & Mann (2000) Nature 405,

9 Large Scale Protein Identification by Mass Spectrometry Mann. EMBO Molecular Medicine (2012) 4,

Use of Shotgun Approaches Now used to fully characterise the human proteome Kuster et al. 2014. Nature 509: 582. Olsen lab. 2017. Cell Systems 4, 587 599.

10 Use of Shotgun Approaches Now used to fully characterise the human proteome Kuster et al Nature 509: 582. Olsen lab Cell Systems 4, ,200 proteins. But Infers the presence of proteins but can not confirm original size Difficult to understand variety of splice forms Difficult to understand the variety of PTM forms

11 Protein Expression Analysis using Stable Isotopes There are stable isotopes of a number of common atoms e.g. 15 N, 13 C, 2 H. These are heavy isotopes Cells can be grown in media with heavy amino acids. These result in heavy-labelled proteins. A mix of heavy and normal ( light ) proteins, from two samples, can be coanalysed by mass spectrometry. Peptides from these proteins show characteristic mass differences. The heights of the two peaks allow you to compare amounts of a protein present in two samples. light peptide heavy peptide Image: L=proteomics-peptide

12 How to Compare Protein Expression Between Samples: The SILAC Technique SILAC: Stable Isotope Labeling by Amino acids in Cell culture 14 N, 12 C and 1 H 15 N or 13 C or 2 H

The heavy plant Reis et al (2015) NATURE PLANTS VOL 1 MARCH 2015.

N-containing media: 15 NH 4 15 NO 3 and K 15 NO 3 Label swap design with wild type,

13 The heavy plant Reis et al (2015) NATURE PLANTS VOL 1 MARCH light plants grown on normal media: NH 4 NO 3 and KNO 3 heavy plants grown on 15 N-containing media: 15 NH 4 15 NO 3 and K 15 NO 3 Label swap design with wild type, mutants. Leaves from WT and mutants harvested, co-analysed. Quantitation achieved by comparative analysis of peak heights.

14 Differential Display using Tandem Mass Tags (TMT) and Mass Spectrometry Comparison of peak heights measures protein quantity per sample

Conclusions Protein separation Protein identification Protein quantification Two excellent references: Matthias Mann Proteomics for biomedicine: a half completed journey EMBO Molecular

15 Conclusions Protein separation Protein identification Protein quantification Two excellent references: Matthias Mann Proteomics for biomedicine: a half completed journey EMBO Molecular Medicine (2012) 4, A. F. Maarten Altelaar, Javier Munoz & Albert J. R. Heck Next-generation proteomics: towards an integrative view of proteome dynamics Nature Reviews Genetics (2013) 14, 35-48

Quantitative mass spec based proteomics

Quantitative mass spec based proteomics Tuula Nyman Institute of Biotechnology tuula.nyman@helsinki.fi THE PROTEOME The complete protein complement expressed by a genome or by a cell or a tissue type (M.