Peptide Mass Finger-Printing Part II. MALDI-TOF 실험조교: 정혜림 Aging Genomics Proteomics Laboratory (Professor Paik) New Science Building Room #305 jeonghl@proteomix.org

Concept of Mass Spectrometry Mass Spectrometer? Instrument measuring molecular weight (MW) of sample Only picomolar concentrations is required Accuracy of 0.01% of total weight of sample Able to detect amino acid substitution/post-translational modifications

Concept of Mass Spectrometry History of Mass spectrometry 1953 : Quadrupole and the ion trap(W. Paul at H.S. Steinwedel). Nobel Prize to Paul in 1989. 1956 : First GC-MS 1968 : First commercial quadrupole 1975 : First commercial GC-MS 1990s : Explosive growth in biological MS, due to ESI & MALDI 2002 : Nobel Prize to Fenn & Tanaka for ESI & MALDI 2005 : Commercialization of Orbitrap MS

Concept of Mass Spectrometry How does Mass Spectrometer work? Ion Source Mass Analyzer Detector Inlet

Concept of Mass Spectrometry Types of Machines / Techniques High Vacuum System 2. Ion Source 3. Mass Analyzer 4. Detector 5. Data System 1. Inlet HPLC Flow injection Sample plate MALDI ESI FAB LSIMS EI CI Time of flight (TOF) Quadrupole Ion Trap Magnetic Sector FTMS Microchannel plate Photomultiplier Electron multiplier

Concept of MALDI-TOF “Sample Preparation” Inlet Peptide Sample Preparation gel Digest Excise Wash Reconstruction Extract Dry Inlet

“1. Inlet: MALDI-TOF matrix” Concept of MALDI-TOF “1. Inlet: MALDI-TOF matrix”

(Matrix Assisted Laser Desorption Ionization) Concept of MALDI-TOF “2. Ion Source: MALDI” (Matrix Assisted Laser Desorption Ionization) Sample plate Laser hn Ionization is triggered by a laser beam A matrix is used to protect the biomolecule from being destroyed by direct laser beam and to facilitate vaporization and ionization AH+ +20 kV

(Matrix Assisted Laser Desorption Ionization) Concept of MALDI-TOF “2. Ion Source: MALDI” (Matrix Assisted Laser Desorption Ionization)

Question “2. Ion Source: MALDI” (Matrix Assisted Laser Desorption Ionization) Which ion will strike the detector faster? Laser The ions enter the flight tube with the lighter ions traveling faster than the heavier ions. SO, GREEN ION WILL STRIKE THE DETECTOR FASTER!

Concept of MALDI-TOF “3. Mass Analyzer: TOF” (Time Of Flight) H+ +22 kV 1) Ions enter source region, accelerated toward reflectron. Reflectron TOF 2) Ions separate in space based on their relative mass-to-charge (m/z). 0 kV 0 kV 3) Ions reverse path in reflectron. 4) Ions impact detector. Flight time Signal 0 kV 0 kV L+ M+ H+ [Molecular weight] +20 kV amp comp

Concept of MALDI-TOF “3. Mass Analyzer: TOF” (Time Of Flight) Linear TOF Linear TOF is used in larger molecules. *we are going to use reflectron TOF.

Concept of MALDI-TOF “4. Detection” Mass accuracy : How accurate is the mass measurement? Resolution : How well separated are the peaks from each other? Sensitivity : How small an amount can be detected / analyzed?

High resolution means better mass accuracy Concept of MALDI-TOF “4. Detection” Mass measurement accuracy depends on resolution High resolution means better mass accuracy 2000 4000 6000 8000 Counts 2840 2845 2850 2855 Mass (m/z) Resolution = 14200 Resolution = 4500 Resolution =18100 15 ppm error 24 ppm error 55 ppm error

Theoretical MALDI TOF SPECTRUM Concept of MALDI-TOF “4. Detection” Theoretical MALDI TOF SPECTRUM Of ONE PEPTIDE MH+ 10000 20000 30000 40000 149876 Relative Abundance (M+2H)2+ (M+3H)3+ 150000 50000 100000 150000 200000 Mass (m/z)

Concept of MALDI-TOF “4. Detection” One 13C atom “Monoisotopic mass” 149876 Two 13C atoms No 13C atoms (all 12C) Three 13C atoms 150000 m/z We calculate resolution and accuracy with these peaks. Annotations on spectra will be for the monoisotopic peaks only.

Assume these are peaks found at [M+4H]4+ Concept of MALDI-TOF “4. Detection” Assume these are peaks found at [M+4H]4+ How to calculate mass? m/z of monoisotopic peak = 431.73 m/z = 431.73 m/4 = 431.73 m=431.73 x 4=1726.92 You must subtract mass of H+ (1) [ 1726.92] – 4 = 1722.92

Please calculate the mass of this peptide. Question “4. Detection” Theoretical Results (M+3H)3+ 3334 (M+4H)4+ MH+ (M+2H)2+ 2501 (M+5H)5+ 10001 5001 2001 Relative Intensity(%) 2500 5000 10000 Assume this is a result of MALDI-TOF for ONE SINGLE PEPTIDE. Please calculate the mass of this peptide.

Solution “4. Detection” 3334 2501 (M+3H)3+ 10001 (M+4H)4+ 5001 2001 Relative Intensity(%) (M+5H)5+ 2500 5000 10000 H+ H+ H+ H+ H+ [M + 5H]5+ [M + 4H]4+ [M + 3H]3+ [M + 2H]2+ [MH]+ M/Z = 10,005 / 5 M/Z = 2001 M/Z = 10,004 / 4 M/Z = 2501 M/Z = 10,003 / 3 M/Z = 3334 M/Z = 10,002 / 2 M/Z = 5001 M/Z = 10,001 / 1 M/Z = 10,001 The same protein with a molecular weight of 10,000 contains 5, 4, 3, 2, and 1 charges

Tryptic peptide mixture. Every peptide has a basic C-terminus. Concepts of MALDI-TOF “5. Database” N K K R K K K R K K Trypsin K K K R R R Protein R R N C K K R Tryptic peptide mixture. Masses measured by MS. Every peptide has a basic C-terminus. C A protein can be identified in a database by matching masses of a subset of the tryptic peptides against calculated values.

Concepts of MALDI-TOF “5. Database” intact protein peptide fragments enzyme MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVS PFDHSRIKLHQEDNDYINASLIKMEEAQRSYILTQGPLPNTCGHFWEMVW EQKSRGVVMLNRVMEKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIK SYYTVRQLELENLTTQETREILHFHYTTWPDFGVPESPASFLNFLFKVRE SGSLSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVL LEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWKELSHEDLE PPPEHIPPPPRPPKRILEPHNGKCREFFPNHQWVKEETQEDKDCPIKEEK GSPLNAAPYGIESMSQDTEVRSRVVGGSLRGAQAASPAKGEPSLPEKDED HALSYWKPFLVNMCVATVLTAGAYLCYRFLFNSNT

Concepts of MALDI-TOF “5. Database” Gel is identical to Database In Silico Digestion In Gel Digestion 848.3 1272.7 493.2 882.6 2978.3 364.1 948.9 3128.8 3514.2 2837.1 263.9 147.4 1429.7 199.6 142.3 640.8 848.1 1272.5 492.6 883.2 2978.9 812.6 1432.3 3127.1 996.8 702.4 164.9 2748.2 is identical to

Concepts of MALDI-TOF “5. Database”

Desalting method Calibration method Techniques for MALDI-TOF Experimental Methods – Tips Desalting method 1) C18 Zip-Tip: Tip containing immobilized C18 2) TFA: Trifluoroacetic acid 3) Matrix solution: matrix mixed with 50% ACN with water Calibration method 1) Standard solution: Examples include Angiotensin II, ACTH fragment

보고서작성 Tips Format: 실험제목, 실험일자, 제출자 및 공동실험자, 목적, 원리, 시약 및 기자재, 방법, 결과, 고찰, 참고문헌 순서로 써주세요. (실험일자에 요일 꼭 써주세요!) (방법은 실험교본 그대로 말고! 직접 실험한 방법!) (고찰 section에 꼭 필요한 내용: DTT와 IAA 사용하는 이유, desalting method 설명, calibration 하는 이유와 방법 설명, 결과에 대한 설명) DUE DATE: 4월 17일 (월요일 저녁 8시까지) SUBMISSION LOCATION: 과학원 S305 