Mass Spectrometry and Proteomics - Lecture 4 - Matthias Trost Newcastle University

Transcription

1 Mass Spectrometry and Proteomics - Lecture 4 - Matthias Trost Newcastle University matthias.trost@ncl.ac.uk

2 previously Peptide fragmentation Hybrid instruments 117

3 The Building Blocks of Life DNA RNA Proteins Lipids Metabolites Genome Transcriptome Proteome Lipidome Metabolome 118

4 The post-genomic Era The knowledge of genome enables us to predict the proteins that can be generated but not where and when or at what level. We now know the protein sequences but still miss the function for many. Genomics can not take into account alternate splicing and post-translational modifications which are likely responsible for the complexity of eukaryotic life. No biological state exists when all genes are expressed. There is no good correlation between the mrna and protein abundance in a cell at a given time. 119

5 One Genome Different Proteomes 120

6 The Proteome Definition: The entire set of proteins of a subcellular compartment, cell, tissue or organism at a given time under defined conditions. The proteome continually changes in response to external and internal events, such as disease, proliferation, specific metabolic state or therapy. The proteome provides the most accurate portrait of the cellular state because it reflects abundance and post-translational modifications of proteins. 121

7 Quantitative Proteomics What is the relative amount of each of the proteins in my samples? What is the exact absolute amount of each of the proteins in my samples? What are the kinetics of changes in the amounts of each of the proteins in my samples, which result from treatments or changes in growth conditions? Same issues as listed above for each of the post-translational modifications of each of the proteins in my samples. 122

8 Bottom-up vs Top-Down Bottom-up Proteins Shotgun proteomics Separate & digest or Digest & separate Peptides MS, MS/MS database search Mass spectrometers IT/TQ/TOF/Orbitrap... Top-down Proteins Separate, MS, MS/MS, database search/analysis High resolution mass spectrometer Proteins identified 123

9 Top-down proteomics 124

10 Top-down advantages Identification of protein isoforms. 100% protein sequence coverage is possible => proteolytic processing events, and PTMs. de novo sequencing. Big protein masses are more "information rich" thus improving the quality of the information and decreasing false positives. Localization of non-covalently bound ligands is possible. 125

11 Top-down disadvantages Limited sensitivity and throughput. Pure samples are required. Insoluble proteins and big proteins can currently not be analysed. Expensive instrumentation, expert level users. 126

12 Bottom-up advantages Simpler Less sophisticated instrumentation required. Higher throughput More info about proteins with extreme phys.-chem. properties (hydrophobic, Hi/Low MW, acidic/basic) 127

13 Bottom-up disadvantages Confidence in protein ID strongly depends on restriction criteria (subjective; potential bias) Protein ID on few peptides is not very safe. Proteins without tryptic peptides in the right mass window are missed. PTM and isoform information is often lost 128

14 Proteomics experiments Complexity (& cost) Protein ID Quantitative pull-down Quantitative whole proteome Quantitative Post-translational Modifications (PTM) In gel digest In solution digest IP His pull-downs Biotin pulldowns Bio-ID Organellar Total cell Total tissue Body fluids Whole animal Multi-species Phosphorylation Ubiquitin Acetylation Oxidation Glycosylation etc 129

15 Mass spec sample prep 130

16 Cell lysis Cell lysis is required to make proteins accessible to denaturation and proteolytic cleavage. It needs to be fast and efficient to reduce unspecific cleavage through intracellular (lysosomal) proteases and other enzymes (phosphatases, DUBs etc.) Mechanical vs chemical Under reducing conditions we prefer 5 mm TCEP Don t forget inhibitors of proteases (careful we need to trypsinise later!), phosphatases, DUBs 131

17 Cell lysis BE CAREFUL WITH DETERGENTS SUCH AS TRITON (aka the mass spec killer) (Exemption: in-gel digest) 132

18 Cell lysis Urea buffers 8 M Urea, 50 mm Tris, 5 mm TCEP, ph 8.0: Heating >30 C can cause carbamylation of amines Trisbuffers can reduce the risk. Urea can precipitate in cold. Always make fresh! 4% SDS Requires removal by FASP/microspin filters or SP3 method. Note: Trypsin is sensitive to >0.1% detergent or 2M Urea. Thus, sample will require dilution before digestion. 133

19 Reduction & alkylation In order to fully denature proteins and avoid the formation disulphide bonds and oxidation of amino acids, we need to work under reducing conditions. Cell lysis should be performed in reducing conditions (recommended 1-5 mm TCEP). Note that sulphide-based reagents such as DTT etc react with alkylating agents such as Iodoacetamide. High concentrations of iodoacetamide + heat lead to alkylation of primary amines such as lysines. This has the same mass as Gly- Gly-modification! use Tris in buffers to avoid side reactions. 134

20 Proteases For large-scale experiments, proteases need to be specific (trypsin, LysC etc.) For purified proteins, unspecific proteases such as elastase can give high sequence coverage. Not compatible with quantification. 135

21 Proteolysis Chemical methods: Cyanogen Bromide cleavage: C-term to Met, leaves a homoserine lactone at Met Enzymatic: Trypsin C-term to Lys, Arg ph 8.5 Chymotrypsin C-term to Y,F, W, H, L ph 8.5 V8 (Glu-C) C-term to Glu ph 8 Lys-C C-term to Lys ph 8 Arg-C C-term to Arg ph 8 Asp-N N-term to Asp ph 8 Thermolysin N-term to L, I, M, F, W ph 8.5 Lys-N N-term to Lys ph

22 Swaney et alj. Proteome Res Peptide lengths

23 FUS protein 138

24 Usage of several proteases increases identification Protease Trypsin ArgC AspN GluC LysC All Unique peptides CAD ETD Total scans Proteins Percent of ORFs Nonredundant amino acids Nonredundant amino acid proteome coverage (percent) Average protein sequence coverage (percent) Swaney et alj. Proteome Res. 2010

25 Sample clean-up Chromatographic step to clean sample of salts, buffers etc Usually C18, but SAX, SCX also available for removal of detergents. Lucci et al, InTechOpen,

26 SP3 method Proteins are locked to carboxylated beads by ionic interactions, allowing removal of detergents and solvents. Hughes, Krijgsveld et al., Mol Syst Biol. (2014)

27 SP3 protocol Workflow