Basic protein and peptide science for proteomics. Henrik Johansson

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1 Basic protein and peptide science for proteomics Henrik Johansson

2 Proteins are the main actors in the cell Membranes Transport and storage Chemical factories

3 DNA Building proteins Structure

4 Proteins mediate signaling cascades leading to gene regulation

5 Composition of a growing E. coli cell A Voronoi tree diagram. Each polygon area represents the relative fraction of the corresponding constituent in the cell dry mass

6 Central dogma i molecular biology Regulatory mechanisms ~ protein coding genes ~ Protein-coding transcripts Epigenetic modifications Alternative splicing mrna stability Translation efficiency Protein-protein interactions Protein stability Post-translational modifications Protein cleavage Subcellular localization

7 Protein complexity DNA ~ genes Pre-mRNA mrna Exon1 Intron1 Exon2 Intron2 Exon3 Exon1 Exon2 Exon2 Exon3 ~90% of genes have splice variants >2 exons, % have splice variants Protein Protein species PO 4 - Enzymatic cleavage COCH 3 ~300 Post translational modifications

8 Complexity of proteome and tryptic peptides DNA ~ genes Proteins Peptides Database Nr of proteins Ensembl ~ Swissprot ~ (manually curated) Nr of fully tryptic peptides, 6-30 amino acid, ~ COCH 3 Peptides with PTMs PO 4 - ~ peptides (predicted) ~ phospho sites known Oxidation of M etc.

9 General approach for shotgun proteomics

10 Our standard proteomics workflow Samples Extract proteins Reduce, alkylate Tryptic digestion itraq labeling SCX cleaning IPG-IEF to 72 fractions LC MS nlc-ms/ms time MS/MS m/z Reporter area m/z Identification

11 Dynamic range of the proteome Schwänhausser 2011 Nature

12 Why peptides instead of proteins in MS based proteomics? Easier to identify by MS Reverse phase Liquid chromatography (RP-LC) separates peptides better than proteins Most proteins give tryptic peptides that are soluble even if the protein itself is insoluble Peptides fragment effectively to give spectra that can be sequenced. Peptides can be detected at lower levels than proteins LC MS/MS time

13 Assumptions in peptide centric proteomics Protein cleavage into peptides (normally trypsin) is specific. Problematic with multiple Rs or Ks close to each other. Produce missed cleavages, which to some extent can be matched. Peptide fragments are matched (not interpreted) to a complete protein database Protein interference problem. One peptide can match to many proteins. Need good sequence coverage to identify isoforms PEPTIDERDKDAG PEPTIDER PEPTIDERDK mrna Exon1 Exon2 Exon2 Exon3 Protein Peptides

14 Peptide centric proteomics is powerfull Identification of all 4400 expressed genes in yeast (6700 genes in total) de Godoy 2008 Nature

15 Evidence for protein coding genes Evidence for protein coding genes

16 Proteins are composed of amino acids and organized in different levels Generally less than 1000 amino acids Up to 100,000 residues Average 60 kda

17 The 20 common amino acids that build proteins

18 Clustering of amino acids according to their physical-chemical properties

19 The charge on amino acid side chains depends on the ph pi (isoelectric point) - the ph at which a particular molecule carries no net electrical charge

20 Bonds in proteins folding -S-S- Disulfide bond Water molecules are implicated in protein structure and stability the hydrophobic effect

21 What are post-translational modifications? Modification of an amino acid by the covalent linkage of chemicals such as acetyl or a phosphoryl group, and the addition of complex structures such as lipids and carbohydrates The cleavage of a finished translated transcript to a mature form for example the processing of signal or activation peptides as well as the autocatalytic protein processing The cross-linking of amino acids such as cysteine and tyrosine PO 4 - COCH 3 -S-S-

22 Functions and examples of posttranslational modifications

23 Post-translational modifications change the properties of peptides and proteins

24 MS based detection of posttranslational modifications Mass differences between modified and unmodified peptides during the MS-MS fragmentation of peptides whole, modified protein vs non modified Amino acid Monoisotopic Mass PhosphoSerine Serine Addition 79.97

25 Example of MS workflow and how properties of amino acid are used Cation exchange low ph Reverse phase high ph Reverse phase low ph