Application of Proteomics in Biological Research An introduction Jau-Song Yu Department of Cell and Molecular Biology Chang Gung University

The central dogma of life science Transcription 1000 X Amplification Translation 100 X Amplification Gene (DNA) mRNA Protein

Genomics: ---Identification and characterization of genes (gene expression) and their arrangement in chromosomes Proteomics (Functional Genomics): ---Functional analysis of gene products (proteins) Bioinformatics: ---Storage, analysis and manipulation of the information from genomics and proteomics

Human Genome Project (HGP) % sequence of human genome published 16 February 2001 Volume 291 Number 5507 The Human Genome 15 February 2001, Volume 409, Number 6822

PNAS USA 98, 10869–10874 (2001) Global gene expression analysis --- cDNA microarray Breast cancer samples vs. normal tissues

The extent of gene expression (i.e. the amount of mRNA) is only one of the many factors determining the protein function in cells C 2 H 5 PO 4 mRNA stability, alternative splicing, etc. Post-translational Modification of proteins (covalent modification, proteolytic cleavage, activator, inhibitor, etc)

Genomics genes characterization and identification Proteomics functional analysis of gene products Bioimformatics

Proteomics --- Global analysis of hundreds to thousands of proteins in cells or tissues simultaneously (why we need?) How to analyze hundreds to thousands of proteins in cells or tissues simultaneously? ● Separation of proteins on one matrix --- two-dimensional gel electrophoresis ● Identification of separated proteins in a high-throughput way --- biomass spectrometry

2-Dimension Electrophoresis (2-DE) for Protein Separation One of the core technology of proteomics is 2-DE: At present, there is no other technique which is capable of resolving thousands of proteins in one separation procedure.

Isoelectric point (pI): Isoelectric point is the pH of a solution at which the net charge of protein is zero. In electrophoresis there is no motion of the particles in an electric field at the isoelectric point. Net charge pH Isoelectric point NH 3 + COOH NH 3 + COOH pH < pI Net positive charge NH 3 + COO - NH 3 + COO - pH = pI NH 2 COO - NH 2 COO - pH > pI Net negative charge

sample pH 9 - pH 3 + Isoelectric focusing (1 st dimension) General principle and protocol of 2-dimension gel electrophoresis MW pH gradient SDS-PAGE Ampholytes polyacrylamide 2nd dimension

Traditional Equipment for Isoelectric focusing (IEF): Ampholytes polyacrylamide Cathode (-) electrode solution Anode (+) electrode solution

Immobilized pH Gradient (IPG) Polyacrylamide gel Acidic buffering group: Basic buffering group: CH2 - CH-C-NH-R O COO - NH 3 + Acrylamide monomer

Gradient maker plastic support film Production of Immobilized pH Gradient (IPG) strip A C B F E D acidic basic pH 3 pH 10

IPGphor (IEF System) Amersham Pharmacia Biotech Inc. Protein IEF Cell Bio-Rad Laboratories Equipment for Isoelectric focusing (IEF):

Lysis solution: 8M Urea 4% NP-40 or CHAPS 40mM Tris base Sample preparation Cell line Lysis solution Sonication vacuum Lysis solution Centrifugation Measurement of [protein] 2-DE sample

IPG strip rehydration and sample loading 2-DE sample Rehydration solution Rehydration solution: 8M Urea 2% NP-40 or CHAPS 2% IPG buffer (Ampholyte) 0.28% DTT Trace Bromophenol blue IPG strip holder Position the IPG strip

IPG strip rehydration and sample loading Strip holder Cathode (-) electrode Anode (+) electrode 30 voltage 12hr

First dimension: Isoelectric focusing 1. Place electrode pads (?) V step-n-hold 1.5hr V step-n-hold 1.5hr V gradient 1500vhr V gradient (?) 36000vhr Time Voltage Holder cover IPG strip Electrode pads

Second dimension: SDS-PAGE SDS equilibration SDS-PAGE SDS equilibration buffer 50 mM Tris-HCl 6 M Urea 30% Glycerol 2% SDS Trace Bromophenol SDS SDS-PAGE 0.5% agarose in running buffer SDS-PAGE Marker in paper IPG strip

Detection of proteins separated on gels --- Protocol of silver stain: 50% methanol 25% acetic acid 4hr ddH 2 O x 3 times 30min/time 0.004% DTT solution 30min 0.1% AgNO 3 30min ddH 2 O 30 sec 3% Na 2 CO % formaldehyde 2.3M citric acid 5% acetic acid 25% methanol

2-DE separation of soluble E. coli proteins

For cancer study ~ Normal cells Tumor cells SDS-PAGE isoelectrofocusing Laser-captured microdissector (LCM) (?????) Clinical specimensCryostat 2D gel electrophoresis Immage system

Identification of 2-DE-separated proteins in a high-throughput way using biomass spectrometry MALDI TOF/TOF MSLC/MS n

What is a mass spectrometer and what does it do? Gary Siuzdak (1996) Mass Spectrometry for Biotechnology, Academic Press

Analogy between mass analysis and the dispersion of light

Components of a mass spectrometer

MALDI-TOF MS (Matrix-assisted laser desorption/ionization-Time of flight ) ( 基質輔助雷射脫附游離 - 飛行時間質譜儀 ) Target plate M/Z Time of Flight Laser First detector Second detectorReflectorTarget plate

MALDI matrix # A nonvolatile solid material that absorbs the laser radiation resulting in the vaporization of the matrix and sample embedded in the matrix. #The matrix also serves to minimize sample damage from the laser radiation by absorbing most of the incident energy and the matrix is believed to facilitate the ionization process.

Matrix-assisted laser desorption/ionization source

Mass Analyzer-Time of Flight (TOF) Kinetic Energy = ½ mv 2 v = (2KE/m) 1/2 m/z

Sensitivity of MALDI-TOF MS ~10 fg g/mole x 5 x mole = 6.74 x 10 –15 g

How to identify 2-DE-separated proteins by MALDI-TOF MS? Linking between genomics/bioinformatics/proteomics Normal cells Tumor cells SDS-PAGE isoelectrofocusing Laser-captured microdissector (LCM) (?????) Clinical specimensCryostat 2D gel electrophoresis Immage system

(?????) MALDI-TOF MS analysis Digested by trypsin (Lys, Arg) Database search/mapping Protein identified (100%?) (621, 754, 778, 835, 1204,, 1398, 1476, 1582) (664, 711, 735, 904, 1079, 1188, 1438) (602, 755, 974, 1166, 1244, 1374) (854, 931, 935, 1021, 1067, 1184, 1386, 1438) (Masses of tryptic peptides are predictable from gene sequence databases) (621, 778, 835, 1204,, 1398, 1582) (735, 904, 1079, 1188, 1438) (755, 974, 1244, 1374) (854, 935, 1021, 1067, 1184, 1386, 1438) (M/Z)

pH310 a b c (B) An example ~ Identification of specific proteins purified from pig brain (A)

(d 2 ) (a 1 ) (b 1 ) (b 3 ) MALDI-TOF analysis of tryptic fingerprint from the proteins purified from pig brain (c 2 )

Data base search for the purified protein from pig brain (c 2 )

MSYQGKKNIP RITSDRLLIK GGKIVNDDQS FYADIYMEDG LIKQIGENLI VPGGVKTIEA HSRMVIPGGI DVHTRFQMPD QGMTSADDFF QGTKAALAGG TTMIIDHVVP EPGTSLLAAF DQWREWADSK SCCDYSLHVD ISEWHKGIQE EMEALVKDHG VNSFLVYMAF KDRFQLTDCQ IYEVLSVIRD IGAIAQVHAE NGDIIAEEQQ RILDLGITGP EGHVLSRPEE VEAEAVNRAI TIANQTNCPL YITKVMSKSS AEVIAQARKK GTVVYGEPIT ASLGTDGSHY WSKNWAKAAA FVTSPPLSPD PTTPDFLNSL LSCGDLQVTG SAHCTFNTAQ KAVGKDNFTL IPEGTNGTEE RMSVIWDKAV VTGKMDENQF VAVTSTNAAK VFNLYPRKGR IAVGSDADLV IWDPDSVKTI SAKTHNSSLE YNIFEGMECR GSPLVVISQG KIVLEDGTLH VTEGSGRYIP RKPFPDFVYK RIKARSRLAE LRGVPRGLYD GPVCEVSVTP KTVTPASSAK TSPAKQQAPP VRNLHQSGFS LSGAQIDDNI PRRTTQRIVA PPGGRANITS LG *908.4 da *2169.1da *pI~5.95 Collapsin Response Mediator Protein-2 (CRMP-2, human)

Proteomics solution IEF SDS-PAGE

Direct identification of the amino acid sequence of peptides by tandem mass spectrometry

Amino acid sequence analysis by MS - an example

Press Release: The Nobel Prize in Chemistry October 2002 The Royal Swedish Academy of Sciences has decided to award the Nobel Prize in Chemistry for 2002 ” for the development of methods for identification and structure analyses of biological macromolecules ” with one half jointly to John B. Fenn Virginia Commonwealth University, Richmond, USA, and Koichi Tanaka Shimadzu Corp., Kyoto, Japan ” for their development of soft desorption ionisation methods for mass spectrometric analyses of biological macromolecules ” and the other half to Kurt W ü thrich Swiss Federal Institute of Technology (ETH), Z ü rich, Switzerland and The Scripps Research Institute, La Jolla, USA ” for his development of nuclear magnetic resonance spectroscopy for determining the three-dimensional structure of biological macromolecules in solution ”. Revolutionary analytical methods for biomolecules This year ’ s Nobel Prize in Chemistry concerns powerful analytical methods for studying biological macromolecules, for example proteins. The possibility of analysing proteins in detail has led to increased understanding of the processes of life. Researchers can now rapidly and simply reveal what different proteins a sample contains. They can also determine three- dimensional pictures showing what protein molecules look like in solution and can then understand their function in the cell. The methods have revolutionised the development of new pharmaceuticals. Promising applications are also being reported in other areas, for example foodstuff control and early diagnosis of breast cancer and prostate cancer. Mass spectrometry is a very important analytical method used in practically all chemistry laboratories the world over. Previously only fairly small molecules could be identified, but John B. Fenn and Koichi Tanaka have developed methods that make it possible to analyse biological macromolecules as well. In the method that John B. Fenn published in 1988, electrospray ionisation (ESI), charged droplets of protein solution are produced which shrink as the water evaporates. Eventually freely hovering protein ions remain. Their masses may be determined by setting them in motion and measuring their time of flight over a known distance. At the same time Koichi Tanaka introduced a different technique for causing the proteins to hover freely, soft laser desorption. A laserpulse hits the sample, which is “ blasted ” into small bits so that the molecules are released. The other half of the Prize rewards the further development of another favourite method among chemists, nuclear magnetic resonance, NMR. NMR gives information on the three-dimensional structure and dynamics of the molecules. Through his work at the beginning of the 1980s Kurt W ü thrich has made it possible to use NMR on proteins. He developed a general method of systematically assigning certain fixed points in the protein molecule, and also a principle for determining the distances between these. Using the distances, he was able to calculate the three-dimensional structure of the protein. The advantage of NMR is that proteins can be studied in solution, i.e. an environment similar to that in the living cell.

The Nobel Prize in Chemistry for 2002 is to be shared between scientists working on two very important methods of chemical analysis applied to biological macromolecules: mass spectrometry (MS) and nuclear magnetic resonance (NMR). Laureates John B. Fenn, Koichi Tanaka (MS) and Kurt Wuthrich (NMR) have pioneered the successful application of their techniques to biological macromolecules. Biological macromolecules are the main actors in the makeup of life whether expressed in prospering diversity or in threatening disease. To understand biology and medicine at molecular level where the identity, functional characteristics, structural architecture and specific interactions of biomolecules are the basis of life, we need to visualize the activity and interplay of large macromolecules such as proteins. To study, or analyse, the protein molecules, principles for their separation and determination of their individual characteristics had to be developed. Two of the most important chemical techniques used today for the analysis of biomolecules are mass spectrometry (MS) and nuclear magnetic resonance (NMR), the subjects of this year’s Nobel Prize award.

Bruker’s movie for MALDI-TOF Mass Spectrometry

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