Strategies for Characterizing Proteins in Proteomics Research

Transcription

1 Strategies for Characterizing Proteins in Proteomics Research Joseph A. Loo Department of Biological Chemistry David Geffen School of Medicine Department of Chemistry and Biochemistry University of California Los Angeles, CA USA

2 Approaches for Protein Identification What is this protein? Molecular weight Isoelectric point (charge) Amino acid composition Other physical/chemical characteristics Partial or complete amino acid sequence Edman (N-terminal sequence) - if N-term. not blocked C-terminal sequence - not commonly performed Mass spectrometry-measured information

3 Electrospray Ionization (ESI) ESI highly charge droplets MS mass/charge (m/z( m/z) Multiple charging More charges for larger molecules MW range > 150 kda Liquid introduction of analyte Interface with liquid separation methods, e.g. liquid chromatography Tandem mass spectrometry (MS/MS) for protein sequencing

4 ESI-MS of Large Proteins distribution of multiply charged molecules (M+15H) (M+14H) (M+16H) 16+ (M+13H) mass m/z ESI-MS (Q-TOF) ph 7.5

5 Matrix-assisted assisted Laser Desorption/Ionization (MALDI) Time-of of-flight (TOF) Analyzer MALDI high voltage v 3 m 3 v 2 m 2 v 1 m 1 detector laser sample drift region m 1 m 2 m 3

6 MALDI Mass Spectrometry of Large Proteins 100 MALDI-MS MS of rat MVP (M+H) + % Intensity (M+2H) m/z

7 Approaches for Protein Sequencing and Identification Top Down MS/MS MIRERICACVLALGMLTGFTHAFGSKDAAADGKPLVVTTIGMIADAVKNIAQGDVHLKGLMGP GVDPHLYTATAGDVEWLGNADLILYNGLHLETKMGEVFSKLRGSRLVVAVSETIPVSQRLSLE EAEFDPHVWFDVKLWSYSVKAVYESLCKLLPGKTREFTQRYQAYQQQLDKLDAYVRRKAQS LPAERRVLVTAHDAFGYFSRAYGFEVKGLQGVSTASEASAHDMQELAAFIAQRKLPAIFIESSI PHKNVEALRDAVQARGHVVQIGGELFSDAMGDAGTSEGTYVGMVTHNIDTIVAALAR Enzymatic or chemical degradation MS/MS Bottom Up

8 Protein Identification by Mass Spectrometry 2-D Gel Electrophoresis MW x Excise separated protein spots pi 1547 In-gel trypsin digest Peptide mass fingerprint by MALDI-TOF or LC-ESI-MS. Additional sequence information can be obtained by MS/MS Recover tryptic peptides m/z Protein identification by searching proteomic or genomic databases

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10 Quadrupole - Time-of of-flight (QTOF) MALDI ESI Q0 Q1 Q2 select precursor ion of interest tandem mass spectrometry MS/MS fragment precursor ion detect resulting product ions

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12 De Novo Protein Sequencing of Heat Resistant Proteins Found in Sacred Lotus Lotus, Nelumbo nucifera, has the distinction of having produced the longest living seed, 1300 yr Seed longevity is attributable to its Fruit coat s impermeability to O 2 and water Rapid adaptability to stress Genome is not known de novo protein sequencing Jane Shen-Miller, Kerry Wooding, Yongming Xie

13 SDS-PAGE Lotus protein (>400 yr) boiled embryo extracts 97 kda 64 kda 51 kda 39 kda 28 kda 19 kda 14 kda Heat hardy proteins are soluble in >100 C enable draught protection Lotus Fe-SOD (oxidative repair) retains 65% activity after 1 hr boiling mass spectrometry and de novo sequencing to identify heat stable proteins

14 Isotope Labeling as an Aid to De Novo Sequencing Trypsin Digestion in H 2 18 O (Arg, Lys) R 1 R 2 R 3 R 4...NH-CH CH-CO-NH-CH-CO-NH-CH-CO-NH-CH-COOHCOOH Trypsin / H 16 2 O / H 18 2 O R 1 R 2 NH 2 -CH-CO-NH-CH-CO- 16 OH R 1 R 2 NH 2 -CH-CO-NH-CH-CO- 18 OH 16 O or 18 O Aids interpretation of MS/MS spectra Distinguish b-ions (from N- terminus) and y-ions (from C- terminus): 2 Da shift

15 MALDI-QqTOF Tryptic Peptide Map trypsin digestion performed in H 2 16 O or H 2 18 O Boiled Embryo Extract 45 kda Band H 16 2 O m/z H O m/z

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17 Enzyme and Chemical Specificity for Proteolysis Arg/Lys - XX Lys - XX Arg - XX Asp/Glu - XX XX - Asp Met - XX Trypsin Lys-C Arg-C V-8 8 protease Asp-N CNBr

18 New Chemistries for Proteomics: Chemical Modification of Cysteine (Cys-specific specific cleavage) N H 2 Br 2-bromoethyl amine Lindley, H. Nature,, 1956, 178,, 647. OH - HS NH O Cysteine H 2 N S N H O Digest with trypsin to cleave after Lys, Arg, Cys S-aminoethyl cysteine Thevis and Loo J. Proteome Res.,, 2003

19 Chemical Modification of Lysine Block Lys-cleavage H 2 N O NH + C H 3 O O CH 3 Lysine Acetic anhydride MeOH H 3 C O H N NH O Digest with trypsin to cleave after Arg, Cys Digest with Lys-C to cleave after Cys Lysine, acetylated (+ 42 Da)

20 Prior to Digestion: Chemical Modification of Cysteine Cys-specific specific cleavage Ionization enhancement of Cys-peptides N H 2 Br OH - HBr N H 2 S R 2-bromoethyl amine S-aminoethyl cysteine Lindley, H. Nature, 1956, 178,, 647. After Digestion: O CH 3 HN NH 2 O-methylisourea H H + N R 2 N N 2 C S H2 NH 2-bromoethyl amine Hale, J.E. et al. Anal. Biochem. 2000, 287,, 110. H H 2 C S + CH 3 OH R homoarginine derivative (improve MALDI response)

21 Human Serum Albumin In-gel derivatization and trypsin digestion MALDI-MS MS % Intensity m/z

22 Human Serum Albumin In-gel derivatization and trypsin digestion (cleavage after Cys, Arg) DAHKSEVAHR FKDLGEENFK ALVLIAFAQY LQQCPFEDHV KLVNEVTEFA KTCVADESAE NCDKSLHTLF GDKLCTVATL RETYGEMADCR CAKQEPERNE CFLQHKDDNP NLPRLVRPEV LVRPEV DVMCTAFHDN EETFLKKYLY EIARRHPYFY APELLFFAKR YKAAFTECCQ AADKAACLLP KLDELRDEGK ASSAKQRLKC ASLQKFGERA FKAWAVARLS QRFPKAEFAE VSKLVTDLTK VHTECCHGDL CHGDL LECADDRADL AKYICENQDS ISSKLKECCE KPLLEKSHCI I AEVENDEMPA DLPSLAADFV ESKDVCKNYA KNYA EAKDVFLGMF LYEYARRHPD YSVVLLLRLA LA KTYETTLEKC CAAADPHECY Y AKVFDEFKPL VEEPQNLIKQ NCELFEQLGE YKFQNALLVR YTKKVPQVST PTLVEVSRNL GKVGSKCCKH PEAKRMPCAE AE DYLSVVLNQL CVLHEKTPVS C DRVTKCCTES LVNRRPCFSA LEVDETYVPK EFNAETFTFH ADICTLSEKE RQIKKQTALV ELVKHKPKAT KEQLKAVMDD FAAFVEKCCK ADDKETCFAE EGKKLVAASQ AALGL Without homoarginine derivatization In addition, with homoarginine derivatization

23 α-lactalbumin In-gel derivatization and trypsin digestion Homoarginine derivatization EQLTKCEVFR ELKDLKGYGG VSLPEWVCTT FHTSGYDTQA IVQNNDSTEY GLFQINNKIW CKDDQNPHSS NICNISC NISCDKF LDDDLTDDIM CVKKILDKVG INYWLAHKAL CSEKLDQWLC EKL 86% sequence coverage D K F L D D D L T D D I M C 14 (homoarg; ) (M+H) + MALDI-MS/MS, MS/MS, QqTOF y 3 y 4 y 7 y 8 y 1 y 2 b 2 b 3 y 5 y 6 y 9 y13 y 10 y 12

24 Why Bother with Intact Protein Masses? The protein s molecular mass defines the native covalent state of a gene s product, including: Post-translational modifications: Glycosylation, Phosphorylation, Oxidation, Deamidation, Lipoylation, Acetylation, Formylation... Post-transcriptional modifications Alternative splicing, Introns/Exons, Frame-shifts, Sequencing Errors Simple confirmation of suspected id s Spotlight the presence of post-translational modifications The fragmentation pattern from proteins can generate sufficient information for identification from sequence databases, particularly when combined with accurate mass measurements of both the intact molecule and its product ions.

25 Combining Gel Electrophoreisis with Mass Spectrometry Building better tools Profiling proteins faster in an automated fashion

26 PAGE-MALDI MALDI-MS: MS: The Virtual 2D Gel Method Potential for automated, high-speed proteomic analysis Rachel Loo

27 Virtual 2-D 2 D Gel Electrophoresis (Analytical Chemistry 73, 4063 (2001)) Identify proteins based on MW and pi Create a database of intact protein masses and provide a direct link to all data produced now and in the future via classical 2D gels Identify proteome-wide posttranslational modifications

28 In-Source Decay (ISD) for Protein Sequencing Peptides and large proteins can be fragmented by ISD Fragmentation occurs in the MALDI ion source not generally well controlled Reflectron TOF not necessary (linear TOF sufficient to measure product ions cut R P F E E D P I T Complete sequence information not present, but extensive stretches of sequence from the N- and/or C-termini observed P E P P E P T P E P T I...TIDE... A G M E N T P E P T I D P E P T I D E

29 Protein ISD from Isoelectric Focusing Gel Counts ptsh 2+ mete S Y W AG N m/z 24 SYWAGNSTREELLAVGRELRARHWDQQKQAGIDLLPVGDFAWYD 67 Counts N-terminal c-ion series S T R E E I/LI/L G R V E I/L R A R H W K/Q D K/Q K/Q K/Q AGIDI/L LP VG D muli F A W Y D Ogorzalek Loo, RR, Cavalcoli, JD, VanBogelen, RA, Mitchell, C, Loo, JA, Moldover, B., and Andrews, PC, Anal. Chem. 73, 4063 (2001). m/z

30 Top-down sequencing of proteins by ESI-MS/MS 100 MW HIV Integrase Catalytic Core (F185K) 827 % m/z

31 Integrase: MS/MS (M+19H) b y % % m/z 764 b y y 2 y 3 y 4 y 5 y 6 y 7 complementary ion pair b Ile -Pro y m/z

32 MS/MS b (m/z 764) b MS/MS/MS or MS 3 can be performed to obtain further sequence information b b b % 1093 b m/z

33 ESI-MS/MS of GroES (97 amino acids) MS/MS yields direct sequence information for the C-terminal 15 amino acid residues. 833 b GroES (Chaperonin-10, MW 10435) b n , n = MS 2 (M+12H) b Difficult to predict fragmentation sites for intact proteins y 5 y y y y y m/z

34 Peptide fragmentation in nozzle-skimmer region of ESI interface Often called nozzleskimmer dissociation Fragmentation can be controlled by adjustment of nozzle-skimmer voltage Low energy CAD peptide fragmentation in atmosphere / vacuum interface Useful for obtaining sequence information for relatively pure samples without the need for a tandem mass spectrometer

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37 Mass Spectrometry as a Tool for Protein Crystallography (see Cohen & Chait, Annu. Rev. Biophys. Biomol. Struct. 2001) Construct Verification Domain Elucidation Define folding domains Improve chances to generate high quality diffracting crystals Limited proteolysis cleaves flexible chains, leaving a more compact folding domain

38 ESI-MS of Intact Protein: ~ 59 kda 3 phosphorylation sites? MW MW Da ESI-MS % m/z

39 Limited Proteolysis for Domain Elucidation ESI-MS Trypsinolysis (15 min) Where are the cleavage sites? Da mass

40 MW MS/MS of Intact Proteins Complex spectra Confirm primary sequence Confirm termini, posttranslational modifications MW (M+18H) 18+ (M+18H) 18+ y-ions b-ions m/z

41 MS/MS of (M+18H) 18+ y-ions similar, but shifted by 80 Da same C-termini, C but phosphorylated MW residues (M+18H) MW residues phospho-site site (M+18H) 18+ y 14+ n y 15+ n y 13+ n m/z

42 Based on homology modeling and limited proteolysis... ESI-MS Trypsinolysis (15 min) (1-150) 150) (1-169) 169) (1-169) 169)-P (1-171) 171)-P ( ) 508)-P mass

43 Conclusions Bottom-up and top-down approaches for protein characterization are complementary Bottom-up methods are robust and lead to efficient processes for identifying proteins Top-down methods are not mature, both experimentally and computationally. Protein fragmentation is not predictable (yet). However, information on protein processing and modifications may be flagged by the top-down approach.