Novel Proteomics Techniques and Bioinformatics 2018/5/2 蛋白質體學 Proteomics 2016 Novel Proteomics Techniques and Bioinformatics 陳威戎 2016. 12. 05

Novel Proteomics Techniques 2018/5/2 1. SELDI protein chips 2. Multiple Dimensional Liquid Chromatography, MDLC 3. Quantitative Proteomics (1) Stable-Isotope Labeling, SILAC (2) Isotope Coded Affinity Tags Technology, ICAT (3) Isobaric Tag for Relative and Absolute Quantitation, iTRAQ 4. Bioinformatics for proteomics

1. SELDI-TOF MS (Ciphergen) 2018/5/2 Surface Enchanced Laser Desorption / Ionization 表面 修飾 雷射 脫附 離子化

SELDI protein chips Surface Enchanced Laser Desorption / Ionization 2018/5/2 Surface Enchanced Laser Desorption / Ionization Protein chips + MALDI-TOF based instrument 表面 修飾 雷射 脫附 離子化 Different chromatographic surfaces (e. g. anion exchanger, cation exchanger, reverse phase)

SELDI protein chips Chemical Surfaces Biochemical Surfaces Hydrophobic Ionic IMAC-3 Mixed Biochemical Surfaces          Antibody DNA Enzyme Receptor Drug

SELDI protein chips Protein chips 2018/5/2 SELDI protein chips SELDI – surface enhanced laser desorption/ ionization Five surface types of 8 array ProteinChips® The Protein Chip Arrays distinguish this technology from other mass spectrometry-based systems. The Arrays provide a variety of surface chemistries for researchers to optimize protein capture and analysis. The chemistries include classical chromatographic surfaces such as hydrophobic for reversed-phase capture, cation-and anion exchange surfaced, immobilized metal affinity capture (IMAC) for capturing metal-binding proteins, and pre-activated surfaces to investigate antibody-antigen, DNA-protein, receptor-ligand, etc. Protein chips

SELDI 2018/5/2

SELDI Protein Chip- suitable for biomarker discovery

SELDI-TOF, Biomarker discovery Animation

Comparative MS profiles among patients and normal control

Comparative MS profiles among patients and normal control

2. Multiple Dimensional Liquid Chromatography, MDLC

2. Multiple Dimensional Liquid Chromatography, MDLC

2. Multiple Dimensional Liquid Chromatography, MDLC

2’. Multi-Dimensional LC-MS/MS

2’. Multi-Dimensional LC-MS/MS

3. Quantitative Proteomics: detection of dynamic changes in tissue/cells Stable-isotope labeling with amino acids in cell culture (SILAC)

Stable-isotope labeling with amino acids in cell culture (SILAC)

Stable-isotope labeling with amino acids in cell culture (SILAC)

Stable-isotope labeling with amino acids in cell culture (SILAC)

Stable-isotope labeling with amino acids in cell culture (SILAC)

Drawbacks: The method does not allow for the analysis of proteins directly from tissue. The stable-isotope enriched media are costly and may themselves affect cellular growth and protein production. The increase in nominal mass because of stable-isotope incorporation is not known until the sequence is determined.

Isotope Coded Affinity Tags Technology, ICAT

Isotope Coded Affinity Tags Technology, ICAT

Isotope Coded Affinity Tags Technology, ICAT

Advantages: Disadvantages: The method is compatible with any amount of protein harvested from bodily fluids, cells or tissues under any growth conditions. The alkylation reaction is highly specific and occurs in the presence of salts, detergents, and stabilizers (e.g. SDS, urea, guanidine-HCl). The complexity of the peptide mixture is reduced by isolating only cysteine-containing peptides. The ICAT strategy permits almost any type of biochemical, immunological, or physical fractionation, which makes it compatible with the analysis of low-abundance proteins. Disadvantages: The size of the ICAT label (~500 Da) is a relatively large modification. The method fails for proteins that contain no cysteines.

SILAC vs. ICAT

isobaric Tag for Relative and Absolute Quantitation , iTRAQ

isobaric Tag for Relative and Absolute Quantitation , iTRAQ

isobaric Tag for Relative and Absolute Quantitation , iTRAQ

isobaric Tag for Relative and Absolute Quantitation , iTRAQ

isobaric Tag for Relative and Absolute Quantitation , iTRAQ

isobaric Tag for Relative and Absolute Quantitation , iTRAQ

isobaric Tag for Relative and Absolute Quantitation , iTRAQ

isobaric Tag for Relative and Absolute Quantitation , iTRAQ

isobaric Tag for Relative and Absolute Quantitation , iTRAQ Non-gel based technique Uses isotope coded covalent tags. Covalent labeling of the N-terminus and sidechain amines of pepitdes from protein digestions with tags of varying mass. Simultaneously identify and quantify proteins from different sources (multiple sample) in one single experiment. Increases confidence in identification and quantitation from MS/MS spectra by tagging multiple peptides per protein. Increases throughput and confidence in results for protein biomarker discovery studies.

isobaric Tag for Relative and Absolute Quantitation , iTRAQ Two mainly used reagents: 4-plex and 8-plex Pooled and fractionated by nano liquid chromatography and analyzed by tandem mass spectrometry (MS/MS) Expands proteome coverage by labeling all peptides, including those with post-translational modifications (PTMs). Offers a simple workflow without sample fractionation for reduced-complexity samples, such as affinity pull-downs.

MS vs. MS/MS vs. Reporter ions

4. Bioinformatics for proteomics

4. Bioinformatics for proteomics

Mascot Search Results Probability Based Mowse Score Score is - Search title : SampleSetID: 362, AnalysisID: 567, MaldiWellID: 15790, SpectrumID: 17225, Path= \ Man i 102004 New Analysis 1 Database : NCBInr 20040606 (1846720 sequences; 611532004 residues) Timestamp : 20 Oct 2004 at 14:52:50 GMT Top Score : 681 for gi|180570 , creatine kinase [Homo sapiens] Probability Based Mowse Score Score is - 10*Log(P), where P is the probability that the observed match is a random event. Protein scores greater than 75 are significant (p<0.05). 42

Top hits from Mascot Search – there are multiple accession numbers for the same protein 43

Creatine kinase B is the highest scoring protein Match to: gi|21536286 ; Score: 681 Creatine kinase - B [Homo sapiens] Nominal mass (Mr): 42591; Calculated pI value: 5.34 Observed Mass & pI: 43kd, 6.2-6.27 Sequence Coverage: 46% 1 MPFSNSHNAL KLRFPAEDEF PDLSAHNNHM AKVLTPELYA ELRAKSTPSG 51 FTLDDVIQTG VDNPGHPYIM TVGCVAGDEE SYEVFKDLFD PIIEDRHGGY 101 KPSDEHKTDL NPDNLQGGDD LDPNYVLSSR VRTGRSIRGF CLPPHCSRGE 151 RRAIEKLAVE ALSSLDGDLA GRYYALKSMT EAEQQQLIDD HFLFDKPVSP 201 LLSASGMARD WPDARGIWHN DNKTFLVWVN EEDHLRVISM QKGGNMKEVF 251 TRFCTGLTQI ETLFKSKDYE FMWNPHLGYI LTCPSNLGTG LRAGVHIKLP 301 NLGKHEKFSE VLKRLRLQKR GTGGVDTAAV GGVFDVSNAD RLGFSEVELV 351 QMVVDGVKLL IEMEQRLEQG QAIDDLMPAQ K 44

List of useful proteomics websites http://www.fixingproteomics.org/ http://www.ionsource.com/ www.ebi.ac.uk www.proteomecommons.org www.peptideatlas.org http://www.biochem.mpg.de/mann http://ncrr.pnl.gov/software/ http://tools.proteomecenter.org http://www.pil.sdu.dk/ www.proteomesoftware.com www.hprd.org www.expasy.org Click on each link to get familiar