Lecture 3-4.pdf - Lecture 3 DNA Ë RNA Ë Protein Reduction...

This preview shows page 1 - 14 out of 61 pages.

Lecture 3 Discovering protein sequence DNA RNA Protein Reduction of virtual to actual protein sequence is often quite iffy
Image of page 1

Subscribe to view the full document.

Literature Bakhtiar, R., & Tse, F.L. S. (2000) Biological mass spectrometry: a primer. Mutagenesis 15 : 415-430. Lopez, M., et al. (2000) A comparison of silver stain and sypro ruby protein gel stain with respect to protein detection in 2-D gels and identification by protein mass profiling. Electrophoresis 21 , 3673- 3683. Rowley, A., et al. (2000) Applications of protein mass spectrometry in cell biology. Methods 20 , 383-397. van Holde, K.E., Johnson, W.C. & Ho, Shing P. (1998) “Mass Spectrometry” from Principles of Physical Biochemistry. Prentice Hall, NJ. Chapter 5.
Image of page 2
Protein sequencing options I. Peptide mapping with overlaps complete protein sequence II. Partial peptide sequence complete cDNA virtual protein sequence nb: it s cheaper and faster BUT be careful Practical limit: ~30 AA
Image of page 3

Subscribe to view the full document.

Protease toolbox Proteases Most common are Trypsin Lys-C-endo Glu-C-endo Subtilisin example Traditional approach - no sequencing, just specific proteases, separation of pieces & composition Rarely successful !
Image of page 4
Example: Cardioactive peptide 9 amino acids Composition after reductive alkylation & hydrolysis CM: 2cmCys, 2F, N, A, T, P, G AE: 2aeCys, 2F, N, A, T, P, G N-terminus: Pro N-terminus: Blocked CT 1. P,T 2. N, A, cmC, F 3. T, G, cmC, NH 3 Elastase 1. N,cmC, P, F, A 2. T, F, A 3. cmC, NH 3 AET 1. P, F, aeC 2. N, F, A, T, G, aeC, NH 3 Method A. Identify ends: Pro X B. Order peptides according to unique Aas, targets, and overlaps CT: P-F aeT: P-F-C EL: P-F-C-N-A CT: N-A-C-F EL: F-T-G CT: T-G-C-NH 2
Image of page 5

Subscribe to view the full document.

Sequencing: 2 methods Automated Edman degradation Applied to full proteins and peptides Mass spectrometry Ladder Fragmentation Hybrid Edman ms/ms Applied to peptides only: recall accuracy of 0.01% Complete mapping with sequence H 2 N + -HN -NH- H 2 N Up to ~30 cycles
Image of page 6
D NSQTEGHARY PMV W FIKL Elution time min Sample cycle #n Abs 267 nm
Image of page 7

Subscribe to view the full document.

Proteomics protocol unknown Intact masses proteolysis peptide masses Search peptide masses Sequence by ms/ms Confirm Protein identity Search database Additional ms/ms unknown unknown known known
Image of page 8
C-ladder sequencing Mass/charge m/z Abundance Usually z = +1 MALDI TOF ms Carboxy peptidase Side-chain masses p. 148
Image of page 9

Subscribe to view the full document.

Amino Acid Residue Mass Monoisotopic Average Glycine Gly G 57.02147 57.052 Alanine Ala A 71.03712 71.079 Serine Ser S 87.03203 87.078 Proline Pro P 97.05277 97.117 Valine Val V 99.06842 99.133 Threonine Thr T 101.04768 101.105 Cysteine Cys C 103.00919 103.144 Isoleucine Ile I 113.08407 113.160 Leucine Leu L 113.08407 113.160 Asparagine Asn N 114.04293 114.104 Aspartic Acid Asp D 115.02695 115.089 Glutamine Gln Q 128.05858 128.131 Lysine Lys K 128.09497 128.174 Glutamic Acid Glu E 129.04260 129.116 Methionine Met M 131.04049 131.198 Histidine His H 137.05891 137.142 Phenylalanine Phe F 147.06842 147.177 Arginine Arg R 156.10112 156.188 Tyrosine Tyr Y 163.06333 163.170 Tryptophan Try W 186.07932 186.213 Amino acid residue mass
Image of page 10
C-ladder sequencing Example ACTH 7-38 1611.7 val 1708.5 lys 1638.6 val 1938.5 tyr 2102 pro 2198.7 asn 2312.8 gly 2389.6 ala 2440.5 glu 2570.7 asp 2688.2 glu 2815.2 ser 2902.3 ala 2971.8 glu 3102.3 ala 3173.5 phe 3320.2 pro 3417.5 leu 3531.4 glu 3659.7 intact ACTH counts Mass, m/z 147 129 163
Image of page 11

Subscribe to view the full document.

CID: 1 collision per molecule 1 2 3 4 1 2 3 4 y” ions b ions
Image of page 12
b and y” ions N - C - C - || | | | H O H R 1 N - C - C | | | H O H R 2 H- Acylium ion b 2 N - C - C - || | | | H O H R 3 N - C - C - OH || | | | H O H R 4 H- y 2 N - C - C - || | | | H O H R 1 N - C - C - || | | | H O H R 2 N - C - C - || | | | H O H R 3 N - C - C - OH || | | | H O
Image of page 13

Subscribe to view the full document.

Image of page 14

{[ snackBarMessage ]}

Get FREE access by uploading your study materials

Upload your study materials now and get free access to over 25 million documents.

Upload now for FREE access Or pay now for instant access
Christopher Reinemann
"Before using Course Hero my grade was at 78%. By the end of the semester my grade was at 90%. I could not have done it without all the class material I found."
— Christopher R., University of Rhode Island '15, Course Hero Intern

Ask a question for free

What students are saying

  • Left Quote Icon

    As a current student on this bumpy collegiate pathway, I stumbled upon Course Hero, where I can find study resources for nearly all my courses, get online help from tutors 24/7, and even share my old projects, papers, and lecture notes with other students.

    Student Picture

    Kiran Temple University Fox School of Business ‘17, Course Hero Intern

  • Left Quote Icon

    I cannot even describe how much Course Hero helped me this summer. It’s truly become something I can always rely on and help me. In the end, I was not only able to survive summer classes, but I was able to thrive thanks to Course Hero.

    Student Picture

    Dana University of Pennsylvania ‘17, Course Hero Intern

  • Left Quote Icon

    The ability to access any university’s resources through Course Hero proved invaluable in my case. I was behind on Tulane coursework and actually used UCLA’s materials to help me move forward and get everything together on time.

    Student Picture

    Jill Tulane University ‘16, Course Hero Intern