Proteomics Global representation of protein composition, interactions, modifications , and activity in temporal context “High content” implicit

Approaches High resolution separation Arrays Multiplex SDS PAGE 1-2D IEF Multidimensional LC Arrays Protein Antibody Multiplex Luminex Aptamers Mass spectrometry

MS based proteomic work flow Sample Ionize Determine mass Process Process Ions Process Data

Ionization methods for peptides Electrospray ionization (ESI) Matrix assisted laser desorption ionization (MALDI)

Beam mass analysers 2:140 (2003)

Trapping mass analysers Quadrupole ion trap Fourier transform ion cyclotron resonance

Schematic of the LTQ Orbitrap Velos MS instrument with three new hardware implementations. Schematic of the LTQ Orbitrap Velos MS instrument with three new hardware implementations. A, the stacked ring ion guide (S-Lens) increases the ion flux from the electrospray ion source into the instrument by a factor 5–10. B, the dual linear ion trap design enables efficient trapping and activation in the high-pressure cell (left) and fast scanning and detection in the low pressure cell (right). C, the combo C-trap and HCD collision cell with an applied axial field with improved fragment ion extraction and trapping capabilities. Olsen J V et al. Mol Cell Proteomics 2009;8:2759-2769 ©2009 by American Society for Biochemistry and Molecular Biology

QqTOF Layout

Peptide Fragmentation Adv Protein Chemistry & Structural Biology, Vol. 80, 1-44, 2010

Archives of Physiology and Biochemistry, 2009; 115(5): 311–319

Tandem Mass Spectrometry (MSMS) Molecular Systems Biology 2008 4:222

Proteins: Considerations Virtually any source Free from degradation Minimal unintroduced chemical modifications Quantity a few micrograms Concentration mg/ml Free from inhibitory materials (e.g. SDS, salts) Dynamic range of materials

Separation Approaches Separation methods 1-2 D PAGE Multidimensional LC Capillary electrophoresis Affinity Antibody Lectin Substrate cofactor PTM

Gel separated Proteins

2D SDS PAGE Strengths Limitations fair resolution Meta data Load size and isoelectric point Molecular heterogeneity (PTMs) Limitations Load Solubility Isoelectric point extremes molecular size variability

2D Difference Gel electrophoresis (DIGE) AppliedBiomics.com

Nat. Inst. Aging

iTRAQ—Isobaric Tags for Relative and Absolute Quantification Multiple tag types available from different commercial sources. Numbers tags/kit constantly rising. Adv Protein Chemistry & Structural Biology, Vol. 80, 1-44, 2010

Comparison of SILAC and iTRAQ Basis of labelling Biosynthetic Chemical Sample labelling Simple Labour intensive Requirements Active metabolism Proteins Defined media Multiplexing Yes Signal Diluted Single ID Inference some times Confirmed Potential for Bias low higher Reutilisation Not applicable Cellular capacity

Induction of Podocalyxin in EMT Log2ratio Frequency Control TGF Beta Control + TGFβ1 - TGFβ1 72h Combine SDS PAGE LC MS/MS TGF

PTM Analysis 14:35 (2013)

Selected Reaction Monitoring Targeted for selected analytes. Not a discovery approach for protein identification Increases sensitivity 30-50 fold Qualitative or quantitative Offers multiplexing capacity 20-30 analytes per run

Selected Reaction Monitoring When antibodies not available Because targeted can ignore high abundance Potential to detect/quantify multiple analytes Demonstrated in this case off by several hundred fold because of interfering antibodies Validation 20-30 different proteins simultaneously in a single sample Used in RA, Renal tspt, biofuels effects of substrate, IP analysis

Activity based protein profiling Inactive enzyme Active enzyme Labelled Unlabelled Carboxyl group of acid hydrogen bonds with histidine - makes electron pair more electronegative Probes available for many categories of enzymes e.g. serine hydrolase, cysteine proteases, ubiquitinase

Marker 2E4A-SH 2E4B-SH LG8A-SH LG8B-SH Normal Lethal Nan Li , Herman S Overkleeft , Bogdan I Florea Activity-based protein profiling: an enabling technology in chemical biology research Current Opinion in Chemical Biology 16:227 2012

2.55 Å crystal structure of SOMAmer SL1025 bound to human IL-6 (form 2 chains A and B). 2.55 Å crystal structure of SOMAmer SL1025 bound to human IL-6 (form 2 chains A and B).A, helices of IL-6 are labeled (A–D) from N to C terminus (term) and are colored magenta (helix A), purple (helix B), blue (helix C), and cyan (helix D). The modified nucleotides of the SOMAmer are colored gold. This color scheme is maintained throughout the figures. B, chemical structures of the C-5 modified deoxyuridines present in SOMAmer SL1025. Gelinas A D et al. J. Biol. Chem. 2014;289:8720-8734 ©2014 by American Society for Biochemistry and Molecular Biology