5 Studying Proteins and Proteomes

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Oklahoma State University *

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3653

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Biology

Date

Oct 30, 2023

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ppt

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21

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Chapter 5 Readings: 5.1, 5.2A-D, 5.3D and E
Proteins and Proteomes The proteome of an organism or tissue or cell 1s a complete list of all proteins at a specific time or environment. The proteome 1s continuously changing. To study a protein or the proteome, one may want to: I. Identify a protein or proteins II. Purity the protein III. Detect i1t 1n a sample IV. See its 3D structure
Identification of a Protein 1 2 3 1 Normal Cells = 2 Cancer Cells —- 3 MW Marker e o = == = ., - ))
SDS Polyacrylamide Gel SDS-PAGE, or SDS polyacrylamide gel electrophoresis, separates proteins based on size. L 1 molecules with net charge (+ or -) can move through liquids, solids or semi- solids 1n an electrical field. (A) (B) ~Q c Q . 5 Haoa’, | Mixture of D2 o 0% macro- o9 < v | molecules Electrophoresis & . . ~ _— (-‘ Direction >| 609 X O/ of electro- phoresis - Porous gel 0 / 2.9 9059 ") Q o\9 | T o) P Polyacrylamide gels are stained (ie. coomassie) so that we can visualize the proteins!
l. Identification of Proteins Mass Spectrometry An analytical technique used to determine the masses of peptides (or molecules) and their sequences. 3 basic components of all mass spectrometers 1. lon source 2. mass analyzer 3. detector @ Beam splitter ot - poin ionized Trigger (2) Electrical field __\ accelerates ions Laser triggers a clock (3) . Lightestions arrive at the M A L D I -TO F detector first Detector
Protein Sequence: Protein #1 Protein #2 A-V-P-G-K-S-T-V-F-F-R-I-L-L A-I-P-G-S-T-V-F-R-C-H add trypsin Vv Vv A-V-P-G-K (470.274 Da) S—T—V—F—F—R (755.386 Da) A‘I‘P‘G'S‘T‘V‘F‘R (946.513 Da) LL-L (357.252 Da) C-H (258.068 Da) 3 peptide fragments 2 peptide fragments TaBLE 3.3 Specific cleavage of polypeptides Reagent Cleavage site Enzymatic cleavage Trypsin Clostripain Staphylococcal protease Thrombin Chymotrypsin Carboxyl side of lysine and arginine residues Carboxyl side of arginine residues Carboxyl side of aspartate and glutamate residues (glutamate only under certain conditions) Carboxyl side of arginine Carboxyl side of tyrosine, tryptophan, phenylalanine, leucine, and methionine
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