Peptide and Protein Analysis Primary (1 ) structure of a peptide or protein is the amino acid sequence

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1 n-suppport cyclization FM R n 1) Pd(0), Et 3 i 2) piperidine R R n PyBP R R n F 3 2, 3, F,! anisole anisole! R n 3 R 2 R 2 49 Peptide and Protein Analysis Primary (1 ) structure of a peptide or protein is the amino acid sequence Amino acid analyzer- automated instrument to determine the amino acid content of a peptide or protein. Individual amino acids are separated by hplc, then detected by post-column derivatization peptide -orprotein [] reduce any disulfide bonds liquid chromatography Enzymatic digestion -or- 3, Δ derivatize w/ ninhydrin 3 R 2 individual amino acids Detected w/ UV-vis Different amino acids have different chromatographic mobilities (retention times) 1972 obel Prize in hemistry William tein tanford Moore 50 25

2 Reaction of primary amines with ninhydrin 3 R 2 Intense purple color Amino Acid Analysis hromatogram o, why is it necessary to use a post- rather than pre-column derivatization protocol? Why are there are only 17 AA s in the chromatogram? 51 Fluorescence Detection- less background, greater sensitivity, lower detection limits Absorption spectroscopy- wavelength that light absorbs, moloecules are in an electronically excited state Emission spectroscopy- the excited molecules relax by emission of a photon. Fluorescence- excitation wavelength and emission wavelength are different. Molecule will emit light at longer (lower energy) wavelength than is absorbs

3 Fluorescent tags Dansyl- detected by UV or fluorescence l R Dansyl chloride R 2 PA (o-phthalaldehyde)- detected by fluorescence 3 R 2 R 2 highly fluorescent 53 Reversed-phase (-18) PL Trace 5 pmols amino acids w/ PA,

4 Attomol detection w/ laser induced fluorescence 2 2 R 2 excitation: 488 nm emission: 560 nm 10-3 milli 10-6 micro 10-9 nano pico fempto atto zepto Avagadro s number ysteine vs. ystine 3 [] [] Ellman's Reagent p λ max = 412 nm ε ~ 13,600 mol -1 ml -1 cm

5 Peptide and Protein equences: primary (1 ) structure- amino acid sequence -labeling with anger s reagent: anger s (2,4-dinitrofluorobenzene) reagent reacts with the -terminal amino group and has a diagnostic UV absorbance that is detected after enzymatic digestion and amino acid analysis 2 2 F 3 2 Δ nucleophilic aromatic substitution enzymatic digestion -or- 3, Δ 2 2 R n 3 plus other unlabeled amino acids -terminal amino acid is specifically labeled with a unique UV chromophore 57 -terminal sequencing: arboxypeptidase- enzyme that hydrolyzed amide bonds of a peptide or protein starting from the -termial end (exopeptidase) R 1 arboxypeptidase 3 3 R 2 Zn 2, 2 R 2 peptide has a new -terminal AA 3 derivatize and identify by PL ydrolyze peptide with hydazine ( 2-2 ) 3 R terminal AA is still an amino acid 2 R n 2 All other AA's are converted to the hydrazides 58 29

6 Edman Degradation: chemical method for the sequential cleavage and identification of the amino acids of a peptide, one at a time starting from the -terminus. Reagent: Ph-==, phenylisothiocyanate Ph p then Ph 2 2 -phenylthiohydantoin:! separated by PL,! detected by UV-vis! -1 peptide with a new! -terminal amino acid! (repeat degradation cycle)! 59 Peptide sequencing by Edman degradation: Monitor the appearance of -phenylthiohydantoin over time to get the peptide sequence. Good for peptides up to ~ 25 amino acids long. Longer peptides and proteins must be cut into smaller fragments before Edman sequencing Fluorescent Edman sequencing reagent 2 2 Fluorescein (a common fluorescent dye) Fluorescein Isothiocyanate (a fluorescent Edman reagent) 60 30

7 Enzymatic and chemical cleavage of peptides and proteins at defined sites 3 Enzymatic trypsin: cleaves at the -terminal side of basic residues, Arg, Lys but not is 2 trypsin chymotrypsin: cleaves at the -terminal side of aromatic residues Phe, Tyr, Trp 2 chymotrypsin thermolysin: cleaves at the -terminal side of hydrophobic residues Phe, Trp, Leu 3 R 2 thermolysin hymotrypsin cleavage products Trypsin cleavage products Tyr Arg Asp-Asn-Gln Leu-Lys Gly-Gly-Phe Ile-Arg-Pro-Lys Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp Tyr-Gly-Gly-Phe-Leu-Arg Trp-Asp-Asn-Gln Trypsin: Tyr-Gly-Gly-Phe-Leu-Arg Arg Ile-Arg-Pro-Lys Leu-Lys Trp-Asp-Asn-Gln hymotrypsin: Tyr Gly-Gly-Phe Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp Asp-Asn-Gln 62 31

8 ther ommonly Used Protein Digest Reagents Glu- cleaves to the -terminal side of Glu residues (cleavage at Asp is times slower) Asp- cleaves to the -terminal side of Asp residues and cysteic acid Lys- cleaves to the -terminal side of Lys residues Lys- cleaves to the -terminal side of Lys residues Arg- cleaves to the -terminal side of Arg residues on-specific proteases: pepsin, proteinase K, subtilysin 63 hemical cleavage of peptides and proteins at defined sites yanogen bromide (Br-): cleaves to the -terminal side of methionine residues 3 2 Br R terminal homo-serine 64 32

9 Lawton s Reagent: cleavage at -terminal side of cysteine residues 2Ph Br Lawton's Reagent Lawton's Reagent 3 Ph 2 R 3 _ 2 2 Ph 2 3 Br 2 2 Ph ys is converted to er 65 EPIDERMAL GRWT FATR (EGF)! 2 -A1 ER2 TYR3 PR4 GLY5 Y6 PR7 ER8 ER9 TYR10! AP11 GLY12 TYR13 Y14 LEU15 A16 GLY17 GLY18 VAL19! Y20 MET21 I22 ILE23 GLU24 ER25 LEU26 AP27 ER28! TYR29 TR30 Y31 A32 Y33 VAL34 ILE35 GLY36 TYR37! ER38 GLY39 AP40 ARG41 Y42 GL43 TR44 ARG45 AP46! LEU47 ARG48 TRP49 TRP50 GLU51 LEU52 ARG53-2!! Trypsin hymotrypsin yanogen Bromide Disulfides bridges at:!ys6 - ys20! ys14 - ys31! ys33 - ys42!!!!. ohen et al. J. Biol. hem. 1972, 247, !!!!! 1972, 247, ! 1973, 248, ! 66 33

10 Peptide sequencing by tandem mass spectrometry Ionization: IM (secondary ion mass spectrometry) Time-of-flight (TF) mass spectrometer Methods to get large, polar molecules into the gas phase for M analysis FAB: Fast Atom Bombardment MALDI: Matrix-Assisted Laser Desorption Ionization EI: Electrospray Ionization Mass spectrometry gives mass/charge (m/z) ratio Introduction to Proteomics: Tools for the ew Biology, Liebler, D.., umana Press: Mass spectrometry is a gas phase technique. Peptides (and proteins) are charged, polar, high molecular weight molecules (ions). ow can peptides and proteins be coaxed into the gas phase? Electrospray ionization (EI): analyte is introduced into the mass spectrometer as an aerosol. liquid chromatography or capillary electrophoresis (separate the analytes)! oulombic fission to the mass analyzer

11 MALDI ionization (matrix-assisted laser desorption): analyte is co-crystallized with an organic molecule that has an intense UV absorption. A laser that is tuned to the absorption of the matrix, is pulsed at the MALDI matrix and energy is indirectly transferred to the analyte. to the mass analyzer 2002 obel Prize in hemistry John Fenn (EI) Koichi Tanaka (MALDI) Laser pulse inapinic acid 2 α-cyano-4-hydroxycinnamic acid (A) 2 2,5-dihydroxybenzoic acid (DB) 69 Mass pectrometry (M): measures the mass to charge ratio (m/z) Dalton (Da) or mass unit (u) = units for measuring molecular masses. ne Da. = 1/12 the mass of the 12 atom Monoisotopic mass sum of the exact masses of the most abundant isotope of each element in a molecule Average mass sum of the averaged masses of each element in a molecules, weighted according to isotopic abundance. ominal mass mass calculated using the integer mass of the most abundant isotope for each element (=1, =12, =16, =14, etc.) Isotope Mass atural Abundance % Isotope Mass atural Abundance 31 P

12 Glu-Gly-Val-Asn-Asp-Asn-Glu-Glu-Gly-Phe-Phe-er-Ala-Arg (EGVDEEGFFAR) Monoisotopic Mass (100%) Average Mass , etc. (80.5%) , etc. (37.3%) , etc. (12.7%) , etc. (3.5%) lide adapted from lecture material developed by A. Burlingame,. Guan, and M. Baldwin (UF) entitled Mass pectrometry and Proteomics m/z 71 As the number of atoms in the molecule increases, the pattern of masses due to the presence of isotopes will change m/z lide adapted from lecture material developed by A. Burlingame,. Guan, and M. Baldwin (UF) entitled Mass pectrometry and Proteomics 72 36

13 What does the isotopic distributions tell us? Relative Abundance Relative Abundance m/z m/z lide adapted from lecture material developed at the University of Lund 73 Resolution and Resolving Power (RP): terms used interchangably igh resolution Low resolution m/z The smallest mass difference (ΔM) between peaks such that the valley between them is a specified fraction of the peak height Full Width alf Maximum (FWM): Width of a single peak measured at 50% peak apex. ΔM lide adapted from lecture material developed by A. Burlingame,. Guan, and M. Baldwin (UF) entitled Mass pectrometry and Proteomics 10% 50% ΔM 74 37

14 Mass Accuracy (MA) - the difference between the experimental mass (M exp ) and the theoretical value (M calc ), calculated from elemental composition. MA = M exp M calc M calc M exp = , M calc = (ppm for high resolution M) accuracy = 9.2 ppm igh resolution means better mass accuracy m/z: M: RP=400 (76.8 ppm) RP=800 (32.1 ppm) RP=1600 (16.2 ppm) RP=3200 (5.5 ppm) lide adapted from lecture material developed by A. Burlingame,. Guan, and M. Baldwin (UF) entitled Mass pectrometry and Proteomics m/z m/z m/z m/z 75 Peptide Mass Fingerprinting : Proteins (or peptides) are digested in a predictable way and the masses of the resulting peptide fragments are unique enough to identify the protein. Requires a database of known sequences and search software to compare (score) the experimentally observed masses with the calculated masses in the database. m/z = 1529 ± 1 Da 478 peptide fragments from ± mouse/human genome ± ± ±

15 Many peptides and proteins give multiply charged ions ID: collision induced dissociation [M ] accelerated into M collision cell (e, Ar, Xe) analyze fragments to get sequence ollision of the [M] ion with the gas causes it to fragment, analysis of these fragments ions gives sequence information charge to -terminus 2 a 1 b 1 c 1 a 2 b 2 c 2 a 3 b 3 c 3 x 1 y 1 z 1 R 2 x 2 y 2 z 2 R 4 x 3 y 3 z 3 -terminus charge to R 2 -terminal fragment R 3 2 -terminal fragment 77 Peptide sequencing by tandem mass spectrometry anospray apillary Electrospray Ion ource Q1 ollision ell (Q2) Q3 to the detector elect peptide to be analyzed fragment the peptide Analyze the peptide fragments elect m/z for Q Peptides fragment in a predictable manner charge to b 1 b 2 b -terminus 3 2 R 2 R 4 y 1 y 2 y 3 charge to -terminus m/z 78 39

16 Amino Acids orted by Mass b 1 b 2 b 3 2 R 2 R 4 y 1 y 2 y 3 average exact - -R- Glycine G Alanine A erine Proline P Valine V Threonine T ysteine Isoleucine I Leucine L Asparagine Aspartic Acid D Glutamine Q Lysine K Glutamic Acid E Methionine M istidine Phenylalanine F Arginine Tyrosine Y Tryptophan W ome ambiguities with M sequencing leucine (L) vs isoleucine (I): difficult to distinguish, must look at fragmentation of the sidechain lysine (K, m/z=128.09) vs glutamine (Q, m/z = ) Ac amu's Ac 2 no reaction gly (G) gly (G) = = asn ()= ala (A) gly (G) = = gln (Q) = = lys (K)= gly (G) val (V) = = arg (R) = ala (A) asp (D) = glu (E) gly (G) = = trp (W) = ser () val (V) = = trp (W) =

17 ID b 5 fragment: m/z = or y 6 fragment: m/z = b 1 y b 2 y 9 3 b 3 3 y b 4 3 y 7 2 b 5 y Glu Leu Val Ile er Leu Ile Val Glu er Lys b 6 y 5 b 7 y 4 b 8 y 3 b 9 y 2 b 10 y

18 83 entral Dogma DA mra protein post-translational modifications genome transcriptome proteome 84 42