Protein and Peptide Sequencing by FTMS Susan Martin

Protein and Peptide Sequencing by FT-ICR MS Susan E. Martin University of Virginia, Charlottesville, VA The University of the Sciences in Philadelphia Department of Chemistry & Biochemistry Philadelphia, PA Office phone usip.edu

Peptide Fragmentation O N H R2R2 O H3NH3N R 1 O N H R 4 O N H R 3 OH b y +

Fragmentation of Tryptic Peptide m/z 147 K 1166 L E D E E L F G S y ions b ions % Relative Abundance y2y2 y3y3 y4y4 y5y5 y6y6 y7y7 b3b3 b4b4 b5b5 b8b8 b9b9 [M+2H] 2+ b6b6 b7b7 y9y9 y8y8

Protein Sequencing by Mass Spectrometry PROTEIN SAMPLE DIGESTION HPLC SEPARATION DISSOCIATION SEQUENCE

Advantages of FT-ICR MS for Protein Analysis Ultra high resolution Accurate mass measurement High sensitivity

Advantages of FT-ICR MS for Protein Analysis Ultra high resolution Accurate mass measurement High sensitivity MS n capability 1. Collision activated dissociation 2. IR photodissociation

Collision Activated Dissociation 1. Precursor isolation (SWIFT) 2. Precursor excitation (SORI) 3. Collision with Argon at 1x10 -6 torr 4. Pump out delay (30 s.) 5. Excite and detect

IR Photodissociation 1. Precursor isolation (SWIFT) 2. Single laser pulse (40W cw CO 2 laser) 3. Excite and detect

CAD vs. IRMPD ADVANTAGES OF CAD More efficient fragmentation ADVANTAGES OF IRMPD Fast No blind spots in spectra water loss from b-ions Laser pulse can burn off salts from sample Similar y- and b- ions are produced from either technique.

FTMS Research Project : 1.Obtain a mixture of proteins. 2.Use electrospray ionization to introduce a complex mixture of proteins into FT-ICR. 3.Isolate individual protein ions and dissociate them to generate amino acid sequence information. 4.Use amino acid sequence information to identify proteins from a database.

Traditional Sequencing Strategy PROTEIN SAMPLE DIGESTION HPLC SEPARATION DISSOCIATION SEQUENCE

Goal of FTMS Research PROTEIN SAMPLE DISSOCIATION SEQUENCE

Research Strategy: Proof of Concept Reduce sample complexity, use only a single protein. Determine feasibility of obtaining useful sequence information. –must be consecutive amino acids. –need a string of at least eight amino acids for unique identification.

m/z % Relative Abundance Charge State Distribution for APV-1

b b b [M+14H] 14+ b b y m/z % Relative Abundance APV-1 CAD MS 2 of

Ac-- A M T D X X S A D D X K K A V G A F A D K S K K K X G V M E F F K K H N F S A E K A F H X X D K D R S G F X E E D E X D V K X X A K T E K D S X D R A D P T F G K X K S V D K D G D G K X G V D E F T S X V T V S -- OH V A G APV-1 Product Ions from MS 2 of

m/z % Relative Abundance Charge State Distribution for APV-1

m/z % Abundance y b b b b b [M+13H] 13+ APV-1 CAD MS 2 of

Ac-- A M T D X X S A D D X K K A V G A F A D K S K K K X G V M E F F K K H N F S A E K A F H X X D K D R S G F X E E D E X D V K X X A K T E K D S X D R A D P T F G K X K S V D K D G D G K X G V D E F T S X V T V S -- OH V A G APV-1 Product Ions from MS 2 of

Identifying Proteins with FT-ICR ? ? ? Intact proteins can absorb energy without producing many fragments. Fragments are formed primarily at aspartic acid residues. Not enough sequence information is generated to identify proteins from databases.

O N H R 2 O N H O OH Aspartic Acid Effect on Peptide Fragmentation

Protein Sequencing by Mass Spectrometry PROTEIN SAMPLE DISSOCIATION SEQUENCE DIGESTION HPLC SEPARATION NO!

Use MS 3 capability of FT-ICR MS to obtain protein sequence information.

Ubiquitin CAD MS 2 of b y y y b b b b [M+11H] m/z % Relative Abundance

H - M Q I F V K T L T G K T I T L E V E P Q Q D P P I G E K D Q I K A K V N E I F A G K Q L E D G R T L S D Y N I Q K HO - G G R L R L V L H L S D T R L I E S T Ubiquitin Product Ions from MS 2 of

H - M Q I F V K T L T G K T I T L E V E P Q Q D P P I G E K D Q I K A K V N E I F A G K Q L E D G R T L S D Y N I Q K HO - G G R L R L V L H L S D T R L I E S T + + Ubiquitin Product Ions from MS 3 of

Product ions (from MS 2 ) with greatest abundance are large protein fragments that provide little new information using MS 3. Smaller product ions have insufficient ion abundance for MS 3. Product ions are still prone to aspartic acid effect.

Methyl Esterification of Peptides CH 3 OH + H 2 O H+H+ O N H O OCH 3 N H O N H O OH N H

Product Ions from MS 2 of Methylated Ubiquitin m/z % Relative Abundance b y y y b b b [M+11H] 11+

H - M Q I F V K T L T G K T I T L E V E P Q Q D P P I G E K D Q I K A K V N E I F A G K Q L E D G R T L S D Y N I Q K HO - G G R L R L V L H L S D T R L I E S T Product Ions from MS 2 of Methylated Ubiquitin

m/z % Abundance y y y y y y b Product Ions from MS 3 of Methylated Ubiquitin

H - P Q Q D P P I G E K D Q I K A K V N E I F A G K Q L E D G R T L S D Y N I Q K MeO - G G R L R L V L H L S D T R L I E S T Ubiquitin Product Ions from MS 3 of Methylated Ubiquitin

H - M Q I F V K T L T G K T I T L E V E P Q Q D P P I G E K D Q I K A K V N E I F A G K Q L E D G R T L S D Y N I Q K MeO - G G R L R L V L H L S D T R L I E S T Product Ions from MS 3 of Methylated Ubiquitin

Research Summary Aspartic acid modification may improve protein fragmentation by CAD. Sufficient amino acid sequence information can be obtained using FTMS to retrieve protein identification from a database.

Protein Sequencing by Mass Spectrometry PROTEIN SAMPLE CAD SEQUENCE DIGESTION HPLC SEPARATION NO!

Protein Sequencing by Mass Spectrometry PROTEIN SAMPLE DISSOCIATION SEQUENCE Chemical Modification

Proteomics Study of the PROTEin complement of the genOME. Nonexistent prior to –Progress in genome sequencing research. –Advances in mass spectrometry. Genome is static. Protein expression is dynamic. –The presence of a gene does not guarantee protein expression. –Proteins do the work of the cell.

Goal of Proteomics Research: Compare healthy and diseased tissue. diagnose disease states. develop new drug therapies. Determine effects of new pharmaceuticals. Cell differentiation, cell death. Provide insight into protein function. Identify proteins that are expressed by a cell population

Challenges of Proteomic Research Cell extracts produce very complex mixtures of proteins. Even more complicated mixtures of peptide fragments from enzymatic digestion. Hundreds of peptides co-elute during a single HPLC run How can sequence information be obtained from each peptide ?!?

Proteomic Strategies Use two-dimensional gel separations to reduce sample complexity. Use mass spec. technology suited for collecting a large number of MS/MS spectra. Use database searching algorithms to identify protein sequences. Use peak parking. –Davis, M. T.; Stahl, D. C.; Hefta, S. A.; Lee, T. D. Anal. Chem. 1995, 67, –Martin, S.E.; Shabanowitz, J.; Hunt,* D. F.; Marto, J.A ; Anal. Chem. 2000, 72,

Proteomics and FTMS Advantages: –High sensitivity. –High mass accuracy. Disadvantages: –FTMS software cannot operate ‘on the fly’. –Extremely difficult to identify a precursor and construct and apply a SWIFT isolation waveform. Solution: –Perform the analysis in two sequential runs.

Mixture of Six Standard Proteins ProteinMoelcular Weight (Da) Solution Concentration b-Casein x M Bovine Serum Albumin x M GA3PDH x M Carbonic Anhydrase (II) x M Beta-lactoglobulin x M Cytochrome C x M

Chromatogram of ion current Time (min) Relative Abundance Tryptic peptides derived from digesting a mixture of six proteins

A Single Mass Spectrum m/z Relative Abundance * Asterisk at mass 736 indicates an ion from Cytochrome C protein present in the mixture at 1x 10  17 moles. Approximately 25 ions are co-eluting. Proteins in mixture are present in 1000-fold concentration range

S G F X E E D E X K bnbn ynyn % Abundance y1y1 y2y2 y3y3 y4y4 y5y5 y6y6 y7y7 b 9 / y 8 b8b8 b7b7 b6b6 b5b5 b4b4 b3b3 b2b2 [M+2H] 2+ m/z

Proteomics Results Protein Number of Fragments Found Amino Acids Identified Percent Coverage b-Casein770/ BSA17152/ GAPDH24298/ CA II992/ BLG966/ Cyto C758/

Success with Unknown Mixtures of Proteins from Cells Peptide sequences were obtained from a complex mixture of proteins. The ultra-high mass accuracy improves confidence in protein assignments and decreases search times using computer database searching algorithms. Proteins were identified.

Results Protein Candidate Precursor m/z (observed) Precursor m/z (predicted) Error (Da) Number of product ions present Actin /30 GAPDH /26 Alpha Crystallin /20 P /18

Acknowledgements Doug BeusmannTracie Bishop Jennifer CaldwellRob Christian Scott FicaroErin Field Leslie FrostAndy High Gina KingJarrod Marto Paul RussoBob Settlage Pam ThompsonForest White Professor Donald F. Hunt Dr. Jeffrey Shabanowitz