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Tarun Gheyi SGX Pharmaceuticals, Inc. / NYSGXRC April 14, 2008

Tarun Gheyi SGX Pharmaceuticals, Inc. / NYSGXRC April 14, 2008. Integration of Mass Spectrometry with High-throughput Protein Crystallography. Introduction.

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Tarun Gheyi SGX Pharmaceuticals, Inc. / NYSGXRC April 14, 2008

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  1. Tarun Gheyi SGX Pharmaceuticals, Inc. / NYSGXRC April 14, 2008 Integration of Mass Spectrometry with High-throughput Protein Crystallography

  2. Introduction In an effort to support various groups in the platform (protein production, crystallization and crystallography), we have made our goals simple: • Use as small amount of protein as we can • Provide as much information as possible on the “precious” protein sample • Reduce the analysis time to synchronize with the high-throughput activities in the platform

  3. Introduction • MS: established tool for analysis of bio-molecules in solution. • MALDI-TOF MS (2000 ppm) + SDS-gel electrophoresis • An accurate measure of sample purity • ESI-MS (200 ppm) • Mass accuracy • At SGX, as part of the NYSGXRC activity, MALDI-MS and ESI-MS are routinely used to monitor the quality of proteins prior to initiation of crystallization trials

  4. MS Lab-Instrumentation • LC-ESI-MS (single quad; accurate mass) • MALDI-TOF (Voyager DE-RP; Linear Mode) • MALDI-TOF (Voyager DE-STR; Reflectron) • capLC-ESI-Q-Iontrap (MS/MS analysis)

  5. MS Criteria Intact MS analysis • Mass accuracy: ± 260 Da (~ 2 mutations) If dM > 260 Da then protein characterization by tandem MS is performed to verify that it is intended protein. If yes, then DNA sequencing is done to identify all mutations • Purity: ≥ 80 % If contaminants are present with the intended protein then an extra purification step (Mono Q-column) is included if there is sufficient protein

  6. Intact protein MS analysis-Purity Representative sample that Pass MS status Clone 9252b1BCt9p1, PID 10963, Pool 1

  7. Intact protein MS analysis-Purity Clone 8662a5KWg2h1, PID 10553, Pool 1 Status: Failed Mass Spec

  8. Intact protein MS analysis-Purity Clone 10120g2BSt20p1, PID 11490, Pool 1 Status: “Passed” Mass Spec BUT--------

  9. Intact protein MS analysis-Crystals 46257 Da • MALDI-MS spectrum showing the MW of the protein crystallized • Crystal was washed with the buffer of the crystallization condition to remove non-crystallized protein and PEG • Thin layer technique is used to spot sample on the MALDI plate that usually is more sensitive than other techniques for large biomolecules.

  10. Intact protein MS analysis-SeMet incorporation HY SeMet media @ 60mg/L Standard M9 media-SeMet @60mg/L HY SeMet media @ 90mg/L • Mass Spectrum showing the affect of concentration of SeMet in M9 and HY media

  11. Intact protein MS analysis- SeMet incorporation SeMet @ 60 mg/L SeMet @ 90 mg/L SeMet @ 120 mg/L • Mass Spectrum showing the affect of concentration of SeMet in HY media • Conclusion • Protein with a “High” solubility rating and less than 10 Methionines in its sequence can have nearly full incorporation at 90 mg/L SeMet. • A protein with more than 10 Methionines in its sequence can have nearly full incorporation at a concentration of 120 mg/L of SeMet.

  12. Conclusions-Intact protein MS analysis • Sample purity and identity is routinely monitored on protein samples • If crystals are obtained on a sample where contaminants were also observed with the intended protein, MS analysis of the crystals can be performed (on request basis) to confirm it’s identity. • Percent SeMet incorporation is routinely monitored. • Heterogeneity due to unknown PTMs are routinely monitored and pursued accordingly (Please check poster with title “Bottlenecks/Solutions for the Amidohydrolase Protein Superfamily” for more information)

  13. Tandem Mass Spectrometry (MS/MS) • MS/MS analysis is performed on all protein samples that have mass discrepancies, as observed by ESI-MS, to determine their true identity. • A batch of 15 samples (15 ug each sample) is simultaneously subjected to trypsin digestion (30:1, protein:enzyme) for 14 hrs at 37°C. • MS/MS analysis is performed on the batch of 15 digested samples using using ESI-quadrupole-ion trap mass spectrometer with online capillary-HPLC. • An example as a representative of different sources of mass discrepancies will be discussed further in this presentation.

  14. High Performance Liquid Chromatography (HPLC)-Tandem Mass Spectrometry (MS/MS) HPLC instrument conditions • Instrument: (Agilent Technologies 1100 series). • Flow Rate: 5 uL/min • Column: Zorbax 300SB C-18; 3.5 uM particle size ; 150 X 0.3 mm • Solvent A: 95% H2O, 5% Acetonitrile, 0.1% Formic Acid • Solvent B: 5% H2O, 95% Acetonitrile, 0.1% Formic Acid • Gradient: 0-10 min: 100% A • 10-60 min: 0-100% B • 60-70 min: 100% B • 70-90 min: 100% A

  15. High Performance Liquid Chromatography (HPLC)-Tandem Mass Spectrometry (MS/MS) • MS/MS instrument conditions • Instrument:Finnigan LCQDECA (Thermoquest, San Jose, CA, USA) ion-trap mass analyzer equipped with ESI source. • The experimental conditions were as follows: • Duration of experiment 90 min • Number of scan events 6 • MS mass range 200-2000 Da • Default charge state 2 • Normalization collision energy 35% (of the maximum) • Activation Q 25 eV • Activation time 30 msec • Activating gas Helium • ESI capillary tip voltage 4.20 kV • ESI source temperature 180ºC • Data dependent MS/MS mode is used. • Sequence-specific ions of the amino acids are used to search a non-redundant protein database with the TurboSequest search engine (Bioworks 3.3) to identify proteins. • The amino acid sequence of the identified protein is searched against in-house SGX_Gold database to identify the targets.

  16. MS/MS analysis- Identification of “Mix-ups” • dM of – 8026 Da was observed in Clone 10337p1BCt11p1 PID 14773 Pool 1. • Trypsin digestion and MS/MS analysis followed by a database search identified this protein as clone 9436c1BCt12p1. • Subsequently, the theoretical mass of clone 9436c1BCt12p1 also matched with the observed mass of this clone. • In the similar fashion, a total of 56 protein samples were identified and were associated with the right clones. The MS/MS spectrum of a doubly charged ion at m/z 813.81. The peptide was identified as AWTPAIAVEVLNSVR (MW 626.90 Da) that belongs to clone 9436c.

  17. MS/MS analysis- Adventitious proteolysis • A dM of -2368 Da was observed in Clone 9257a1BCt12p1 PID 13377 Pool 1 and MS/MS analysis identified the tryptic peptides as part of the intended protein. • DNA and glycerol stock sequencing did not identify any mutations or errors and hence the dM observed was attributed to in-cell proteolysis. • Another observation that supported this argument was none of the peptides that were identified covered the C-terminal sequence. • Moreover, by accurate mass measurement the observed mass matched with A[24-426]H. • Using this method a total of 25 protein samples were identified and were associated with right sequence. The MS/MS spectrum of a doubly charged ion at m/z 638.7. The peptide was identified as IWNGYSPLGLR (MW 1275.68 Da) that belongs to clone 9257a.

  18. MS/MS analysis- Molecular Biology Errors • dM of +2150 Da was observed in Clone 11012k2BCt2p1 PID 16672 Pool 1. • MS/MS analysis identified it as an intended clone. • Plasmid and glycerol stock sequencing identified that there was a DNA deletion in the vector His tag causing a frame shift that read through the stop until the next one was encountered ~2150 Da downstream. • Using this method a total of 29 protein samples were identified that were followed by sequencing and rectified sequences were uploaded in LIMS. The MS/MS spectrum of a doubly charged ion at m/z 772.22. The peptide was identified as AM#TLHLLDLSPER (MW 1542.79 Da) that belongs to clone 11012k.

  19. Conclusions-Tandem Mass Spectrometry • In 2007, ~1400 protein samples were analyzed by ESI and MALDI-MS. • Out of these 120 samples of questionable identity were further analyzed by MS/MS. • Using mass spectrometry we have identified the following common problems that can lead to mass discrepancies and number of structures thus benefited from it: • Results indicated that these fit into the above groups as follows: • 1. Clone “mix-up”: 56 of which 8 resulted in structures; • 2. Cloning, etc. artifacts: 29 of which 3 resulted in structures; • 3. Truncation by proteolysis: 25 of which 3 resulted in structures; • 4. Unwanted E. coli contaminant: 10, none of which were pursued for structure determination. • In conclusion, our standard MS quality control analysis procedures contributed substantially to the success of 14 of a total 158 NYSGXRC PDB depositions during calendar 2007 and allowed us to avoid wasting effort on 10 inadvertently purified protein samples.

  20. In the end------- • In an effort to support various groups in the platform (protein production, crystallization and crystallography), we have made our goals simple: • Use as less sample as we can-Never used more than 100ug of sample under study • Provide as much information possible on the “precious” protein sample-Protein purity, Identity, PTM study, crystal analysis, SeMet incorporation, In-gel trypsinization MS/MS analysis, Identification of mix-ups, In-cell proteolysis, Molecular Biology errors etc • Reduce the analysis time to synchronize with the high-throughput activities in the platform-use of high throughput MS approach helps us to reduce analysis time

  21. Acknowledgement • This work was supported by SGX Pharmaceuticals, Inc. and NIH Grant U54 GM074945 (Principal Investigator: Stephen K. Burley)

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