Thermo  Fisher  Scien.fic  –  Annual  Users’  Mee.ng  

ASMS  2011  -­‐  June  5,  2011  

Top Down Proteomics Enabled by the Orbitrap Elite  

Kelleher  Research  Team  Northwestern  University    

Executive Summary

•  Top Down Proteomics: A Full Solution for Protein Isoforms in Discovery Mode

•  Top Down MS: Targeted Analysis of Intact Antibodies

Detailed Summary

•  Top Down MS: Contemporary Context •  Benchmarking the Elite on Protein Standards •  Targeted Top Down MS of Humira Ab •  Use of the Orbitrap Elite for Top Down

Proteomics of Human Cells •  Database Strategies and Informatics for Precision

Proteomics: ProSight Suite of Software

Data from an Orbitrap Elite, equipped with ETD, HCD, CID and SID

Detailed Summary

•  Top Down MS: Contemporary Context •  Benchmarking the Elite on Protein Standards •  Targeted Top Down MS of Humira Ab •  Use of the Orbitrap Elite for Top Down

Proteomics of Human Cells •  Database Strategies and Informatics for Precision

Proteomics: ProSight Suite of Software

Data from an Orbitrap Elite, equipped with ETD, HCD, CID and SID

• Top Down (intact protein)

• Middle Down (endogenous biomarkers, larger peptides >3 kDa)

• Bottom Up (tryptic peptides; classical)

N C

N C

N C

General Strategies of Protein Analysis by Mass Spectrometry

Protein Variability in Higher Proteomes and BioPharma:

The Age of Protein Isoforms

Top Down Proteomics: 2002  

29  September,  2008  

Proteomics using intact proteins as the primary unit of measurement

Detailed Summary

•  Top Down MS: Contemporary Context •  Benchmarking the Elite on Protein Standards •  Targeted Top Down MS of Humira Ab •  Use of the Orbitrap Elite for Top Down

Proteomics of Human Cells •  Database Strategies and Informatics for Precision

Proteomics: ProSight Suite of Software

Orbitrap Technology: Upgrades for Next-Gen. Proteomics

New: •  Generation II Ion Optics

•  New detection system and pre-amplifier

•  Compact HF (3.5 kV)

•  eFT data processing

à  >3.6 X speed increase

à  Orbitrap physics: (m/z)-1/2 scaling of RP

Time (min) 35 40 30 55 50 45 20 25 15

Ubiquitin-9+ R= 154 K

Myoglobin-17+ R= 141 K

Trypsinogen-12+ R= 107 K

952.0 952.2 952.4 952.6 952.8 953.0 953.2 953.4m/z

1999.0 1999.2 1999.4 1999.6 1999.8m/z

997.7 997.8 997.9 998.0 998.1 998.2 998.3 998.4 998.5 998.6m/z

1199.8 1200.0 1200.2 1200.4 1200.6 1200.8 1201.0m/z

Superoxide Dismutase-

13+ R= 132 K

1161.7 1161.8 1161.9 1162.0 1162.1 1162.2 1162.3m/z

Carbonic Anhydrase-25+

R= 115 K

Top Down of the Usual Suspects on Orbitrap Elite

FT  Mass  Spectrometry  

Thermo  7T  LTQ-­‐FT  Ultra  High-­‐resolu>on  Pep>de  Analysis  

Thermo  Velos-­‐Orbitrap  High-­‐resolu>on  Pep>de  Analysis  

Custom  12T  LTQ-­‐FT  Ultra  Top  Down  Protein  Characteriza>on  

Comparison of FT-Orbitrap and FT-ICR: Resolving Power

Detailed Summary

•  Top Down MS: Contemporary Context •  Benchmarking the Elite on Protein Standards •  Targeted Top Down MS of Humira Ab •  Use of the Orbitrap Elite for Top Down

Proteomics of Human Cells •  Database Strategies and Informatics for Precision

Proteomics: ProSight Suite of Software

ETD of intact 150 kDa antibody: Humira IgG

148.0 148.5 149.0Mass, kDa

LTQ Velos Pro Orbitrap MS/MS ETD: fluorenthene, 10-25 ms LC: BioBasic-4 column (10 min gradient) Protein amount: 1 pmol per injection Number of scans summed: up to 1000

ETD of intact 150 kDa antibody: Humira IgG

m/z

ETD: 25 ms

Sequence coverage: ETD of Humira IgG, [M+54H]54+

Light Chain: 46 c-ions, 23 y-ions, 31 z-ions Heavy Chain: 48 c-ions, 21 y-ions, 35 z-ions Total unique cleavage sites: 161

N-acetylglucosamine

mannosefucosegalactoseN-acetylglucosamine

mannosefucosegalactose

Tsybin et al. Poster 557

Monday

Detailed Summary

•  Top Down MS: Contemporary Context •  Benchmarking the Elite on Protein Standards •  Targeted Top Down MS of Humira Ab •  Use of the Orbitrap Elite for Top Down

Proteomics of Human Cells •  Database Strategies and Informatics for Precision

Proteomics: ProSight Suite of Software

Data Acquisition and Processing

Work Flow for Top Down Proteomics

Cell lysate GELFrEE intact protein fractionation

–  

+  

100  kDa  

10  kDa  

High-throughput search using ProSightPC

Online nanoLC-MS/MS on 12 Tesla LTQ-FTMS

10 kDa

25 kDa

(60-90 min separation) (200 – 400 µg)

Lee, Kellie, Kelleher and co-workers, J. Am. Soc. Mass Spectrom. 2009.

Detergent Removal Columns

400 600 800 1000 1200 1400m/z

Orbitrap Elite - Top Down Acquisition Cycle Intact MS1 216K RP

500 ms MS2 108K RP

275 ms Top 3 (transient, reaction time, overhead)

1.5 s Cycle Time

700 800 900 1000 1100 1200m/z

Data Dependent MS/MS

ProSight Search Intact Mass Determination

Fragment Mass Determination

MS1 Injection ~1 ms

MS2 Injection ~50 ms

Intact and Fragment Mass Determination: ProSight HTTD Logic

Durbin, K.; Tran J.T.; Zamdborg, L.; Kelleher, N.L., Proteomics. 2010, 10, 3589-3597.

0 10 20 30 40 50 60 70 80Time (min)

Metrics  From  A  Single  Injec>on  Top  3  HCD  Experiment  

MS/MS  spectra  

Experiments  with  ≥4  Matching  Frag.  

Total  ProSight  ID’s  

(E<0.01)    

Posi.ve  Results  

Unique  Proteins  

Unique  Isoforms  

585   1892   1037   55%   33   78  

1108.2 1108.6 1109.0 1109.4 1109.8m/z538.5 538.7 538.9 539.1 539.3 539.5

m/z

Coiled-­‐coil  domain-­‐containing  protein  72;  E-­‐value  4  E-­‐16  Profilin-­‐1;  E-­‐value  2  E-­‐16  Ribonuclease  UK114,  E-­‐value  7  E-­‐18  

998.0 999.0 1000.0m/z

Scope  of  the  Study  Thus  Far  (H1299  Cells)  

10  

15  

20  

25  

37  

50  

 60  Files  HCD/ETD/CID  DD      

 34  Files    16  HCD  DD  

28  High-­‐High  SID    

18  Files  Med-­‐High-­‐SID      

Database  Searching:    Overall  Results  Ø  ProSight  Experiments:    52081  Ø  Total  Iden>fica>on  Events  (E-­‐value  <0.01):    40779  

Ø  Hits  with  an  annotated  PTM:    33808  

Ø Unique  Swiss-­‐Prot  Accession  Numbers:    501  Ø  Dis.nct  Protein  Isoforms:  1792  Ø Number  of  Alterna>ve  Splice  Events  (var_seq):    32  Ø Number  of  PTMs  (Unique  Sites)  Detected:    1300  

Ø   Phosphoryla>on:    319  Ø   Methyl(x):    86  Ø   Myristoyl-­‐/Palmitoyl-­‐:    3  Ø   Actetyla>on:    406  

Ø   N-­‐terminal  Acetyla>on:    483  Ø   Geranylgeranyla>on:    2  Ø   Hypusine:    1  

Distribu>on  of  Observed  Masses  (kDa)  

Overall  Results  (con>nued)  One  in  a  million  chance  of  obtaining  wrong  

result!  

Hyper  Confident  ID’s  

0  

10  

25  

80  

-­‐log  (E-­‐value)    

•  90  .RAW  Files  •  Frac>ons  <55kDa  20  

15  

5  

m/z

1158.02z=35

1155.66z=35

Example  Iden>fica>on  at  40  kDa  (FT/FT)  

900 1000 1100 1200 1300 1400 1500m/z 800 1000 1200 1400 1600 1800

m/z

MS2  SID  Reduces  the  an>-­‐apopto>c  ac>vity  of  Bcl-­‐xl  and  Bcl-­‐2.    

Δ80  

Retriculon  4B  (Isoform  2):    

@ !@E!@D!@L @D @Q!@S @P!@L!@V!@ !@S!@S!@D!@S!@P!@P!@R!@P!@Q!@P!@A!@F!@K!@Y!@Q!@F!@V!@R!@E!@P!@E!@D!@E!@E!@E!@E!@E!@E!@E!@E!@E!@E!@D!@E!@D!@E!@D!@L!@E!@E!@L!@E!@V!@L!@E!@R!@K!@P!@A!@A!@G!@L!@S!@A!@A!@P!@V P!@T!@A P!@A!@A!@G!@A P!@L!@M!@D!@F!@G!@N!@D!@F!@V!@P!@P!@A!@P!@R!@G!@P!@L!@P!@A!@A!@P!@P!@V!@A!@P!@E!@R!@Q!@P!@S!@W!@D!@P!@S!@P!@V!@S!@S!@T!@V @P!@A!@P!@S!@P!@L!@S!@A!@A!@A!@V!@S!@P!@S!@K!@L!@P!@E!@D!@D!@E!@P!@P!@A!@R!@P!@P!@P!@P!@P!@P!@A!@S!@V!@S!@P!@Q!@A!@E!@P!@V!@W!@T!@P!@P!@A!@P!@A!@P!@A!@A!@P!@P!@S!@T!@P!@A!@A!@P!@K!@R!@R!@G!@S!@S!@G!@S!@V!@V!@V!@D!@L!@L!@Y!@W!@R!@D!@I!@K!@K!@T!@G!@V!@V!@F!@G!@A!@S!@L!@F!@L!@L!@L!@S!@L!@T!@V F!@S!@I!@V!@S!@V!@T!@A!@Y!@I!@A!@L!@A!@L!@L!@S!@V!@T!@I!@S!@F!@R!@I!@Y!@K!@G!@V!@I!@Q!@A!@I!@Q!@K!@S!@D!@E!@G!@H!@P!@F!@R!@A!@Y!@L!@E!@S!@E!@V!@A!@I!@S!@E!@E!@L!@V!@Q!@K!@Y!@S!@N!@S!@A!@L!@G!@H!@V!@N!@C!@T!@I!@K!@E!@L!@R!@R!@L!@F!@L!@V!@D!@D!@L!@V!@D!@S!@L!@K!@F!@A!@V!@L!@M!@W!@V!F!T!Y!V!G!@A!L!F!@N!@G!@L!T!L!L!I!L!A!L!I!S!L!F!S!V!P!V!@I!@Y!@E!@R!@H!@Q!@A!@Q!@I!@D!@H!@Y!@

L!@G!@L!@A!@N!@K!@N!@V!@K!@D!@A!@M!@A!@K!@I!@Q!@A!@K!@I P!@G!@L!@K!@R!@K!@A!@E!

Theore>cal  Mass:  40497.1  Da  

Observed  Mass:  40495.4  Da    Mass  Error:    42  ppm  

Total  Matching  Ions:    34  

* *

Detailed Summary

•  Top Down MS: Contemporary Context •  Benchmarking the Elite on Protein Standards •  Targeted Top Down MS of Humira Ab •  Use of the Orbitrap Elite for Top Down

Proteomics of Human Cells •  Database Strategies and Informatics for

Precision Proteomics: ProSight Suite of Software

Correct Gene Family

Correct Gene

Correct Protein Isoform

Shotgun Annotation Extended to the Whole Human Proteome

Roth, Kelleher, and Team, Mol Cell Proteomics, 2005, 7, 1002-1008.

Update of the UniProtKB / Swiss-Prot

Release Version

“2011_04” (05-Apr-11)

Feature counts for Swiss-Prot (Homo sapiens; April 2011)

20,225 total entries: •  CONFLICT !was found 31853 times.!•  VAR_SEQ !was found 20511 times.!•  VARIANT !was found 64108 times.!•  PEPTIDE !was found 373 times.!•  PROPEP !was found 795 times.!•  SIGNAL !was found 3447 times.!•  CHAIN !was found 20747 times.!•  TRANSIT !was found 486 times.!•  MOD_RES !was found 39539 times.!•  INIT_MET !was found 1478 times.!

Swiss-Prot Entry for HMGA1

ID HMGA1_HUMAN Reviewed; 107 AA. AC P17096; P10910; Q5T6U9; Q9UKB0; DT 01-AUG-1990, integrated into UniProtKB/Swiss-Prot. DT 23-JAN-2007, sequence version 3. DT 05-APR-2011, entry version 140. DE RecName: Full=High mobility group protein HMG-I/HMG-Y; DE Short=HMG-I(Y); DE AltName: Full=High mobility group AT-hook protein 1; DE Short=High mobility group protein A1; DE AltName: Full=High mobility group protein R; GN Name=HMGA1; Synonyms=HMGIY; OS Homo sapiens (Human). OX NCBI_TaxID=9606; FT INIT_MET 1 1 Removed. FT CHAIN 2 107 High mobility group protein HMG-I/HMG-Y. FT MOD_RES 2 2 N-acetylserine (By similarity). FT MOD_RES 15 15 N6-acetyllysine. FT MOD_RES 26 26 Asymmetric dimethylarginine; in isoform HMG-I alternate. FT MOD_RES 26 26 Omega-N-methylarginine; in isoform HMG-I alternate. FT MOD_RES 26 26 Symmetric dimethylarginine; in isoform HMG-I; alternate. FT MOD_RES 36 36 Phosphoserine. FT MOD_RES 39 39 Phosphothreonine. FT MOD_RES 44 44 Phosphoserine. FT MOD_RES 49 49 Phosphoserine. FT MOD_RES 53 53 Phosphothreonine. FT MOD_RES 58 58 Asymmetric dimethylarginine; by PRMT6; alternate. FT MOD_RES 58 58 Omega-N-methylarginine; by PRMT6; alternate. FT MOD_RES 60 60 Asymmetric dimethylarginine; by PRMT6; FT MOD_RES 60 60 Omega-N-methylarginine; by PRMT6; FT MOD_RES 99 99 Phosphoserine; in isoform HMG-I and isoform HMG-Y. FT MOD_RES 102 102 Phosphoserine; by CK; in isoform HMG-I and isoform HMG-Y. FT MOD_RES 103 103 Phosphoserine; by CK; in isoform HMG-I and isoform HMG-Y. FT VAR_SEQ 35 45 Missing (in isoform HMG-Y). FT VAR_SEQ 66 107 NKGAAKTRKTTTTPGRKPRGRPKKLEKEEEEGISQESSEEE FT Q -> KNWRRRKRRASRRSPRRRSSDPCVPPAPHWRSSFLL FT GLDSFAPLPPPPPLPQAHHHHRLWPPPPSSTCALTTTLHST FT PAAAGLPWAEWGAVFPWPQFPAPPAHPRIHTCPPGQG (in FT isoform HMG-R). SQ SEQUENCE 107 AA; 11676 MW; E9C4E3F2200914B8 CRC64; MSESSSKSSQ PLASKQEKDG TEKRGRGRPR KQPPVSPGTA LVGSQKEPSE VPTPKRPRGR PKGSKNKGAA KTRKTTTTPG RKPRGRPKKL EKEEEEGISQ ESSEEEQ //

VAR_SEQ indicates splice variants

MOD_RES indicates post-translational modifications

This entry will produce 3 ProSight

Base Sequences and 32,256 ProSight Protein Forms

Proper Presentation of Human Protein Databases for Mass Spectral Searching

UniProt entries for Homo Sapiens

Features Recognized

Reduce Redundant Sequences

Allow 213 PTMs/SNPs possible per sequence

54190 Base Sequences

20225 Entries

8.5 Million Isoforms

(5 GB)

•  Initial Methionine •  Chain •  Known Modifications •  Sequence Variants •  Conflicts

The Process of “Shotgun Annotation”

(Kelleher Group, JACS, 2004) Allows automated detection of

combinatorial modifications

ProSightPC  Evolu>on  

Windows7  !  X64  !  New  Features  !  

Detailed Summary

•  Top Down MS: Contemporary Context •  Benchmarking the Elite on Protein Standards •  Use of the Orbitrap Elite for Top Down

Proteomics of Human Cells •  Database Strategies and Informatics for Precision

Proteomics: The ProSight Way •  Targeted Top Down MS of Humira Ab

Conclusions ❒ Top Down Proteomics in a robust platform is now possible in the 5-40 kDa regime. ❒ 40-150 kDa: under accelerating development and targeted analysis in this size regime is readily performed now. ❒ The Orbitrap Elite is an ultra-high performance instrument for Top Down on a LC timescale (>3.6 X). Complementarity of fragmentation methods useful for both éI.D.’s and localizing PTMs. ❒ Proteome studies of DNA-damage models and primary cancer/serum samples now underway.

Acknowledgements

•  Iain Mylchreest •  Alexander Makarov •  Michael Senko •  Stevan Horning •  Oliver Lange •  Vlad Zabrouskov •  Andreas Huhmer

•  Eugene Damoc •  Eduard Denisov •  Alain Guiller •  Hartmut Kuipers •  Jens Griep-Raming

The Chemistry of Life Processes Institute

Kelleher  Research  Group  

Proteomics Center @ Northwestern

Dr. Paul Thomas, Associate Director

Dr. Phil Compton, Head of Instrumentation

Mr. Ryan Fellers, Senior Software Engineer

Thank You