Transcript NYSGXRC - PSI Nature Structural Biology Knowlegebase : SBKB

Integration of Mass Spectrometry with Highthroughput Protein Crystallography
Tarun Gheyi
SGX Pharmaceuticals, Inc. / NYSGXRC
April 14, 2008
Introduction
In an effort to support various groups in the
platform (protein production, crystallization and
crystallography), we have made our goals
simple:
1. Use as small amount of protein as we can
2. Provide as much information as possible on the
“precious” protein sample
3.Reduce the analysis time to synchronize with
the high-throughput activities in the platform
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
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)
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 Qcolumn) is included if there is sufficient protein
Intact protein MS analysis-Purity
Representative sample that Pass MS status
Clone 9252b1BCt9p1, PID 10963, Pool 1
Intact protein MS analysis-Purity
Clone 8662a5KWg2h1, PID 10553, Pool 1
Status: Failed Mass Spec
Intact protein MS analysis-Purity
Clone 10120g2BSt20p1, PID 11490, Pool 1
Status: “Passed” Mass Spec BUT--------
Intact protein MS analysis-Crystals
Voyager Spec #1[BP = 21755.4, 3313]
21755. 50
100
90
3313. 0
5465. 09
14508. 48
5392. 18
6118. 95
6000. 40
5887. 88
6307. 21
6256. 99
80
70
46257 Da
10871. 82
8699. 62
43557. 56
% I n ten si ty
60
6899. 81
7003. 27
43417. 57
7310. 80
50
7705. 28
43680. 32
8188. 21
8494. 30
8947. 69
40
21634. 09
43222. 26
10942. 71
30
14581. 00
9801. 75
10181. 93
20
5274. 85
10
0
4999. 0
43119. 13
43045. 66
21584. 50
9328. 68
11133. 43
12660. 39
21516. 99
21443. 86
14643. 01
17406. 26
17160. 28
15999. 4
42928. 51
42727. 85
20966. 08
20000. 01
29130. 45
37493. 45
22898. 83
26999. 8
38000. 2
40949. 46
40240. 60
44059. 74
45685. 28
49000. 6
Mass (m/ z)
• 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.
0
60001. 0
Intact protein MS analysis-SeMet incorporation
Standard M9 media-SeMet @60mg/L
HY SeMet media @ 60mg/L
HY SeMet media @ 90mg/L
• Mass Spectrum showing the affect of concentration of SeMet in
M9 and HY media
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.
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)
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.
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
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
Number of scan events
MS mass range
Default charge state
Normalization collision energy
Activation Q
Activation time
Activating gas
ESI capillary tip voltage
ESI source temperature
90 min
6
200-2000 Da
2
35% (of the maximum)
25 eV
30 msec
Helium
4.20 kV
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.
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.
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.
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.
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.
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
Acknowledgement
• This work was supported by SGX Pharmaceuticals,
Inc. and NIH Grant U54 GM074945
(Principal Investigator: Stephen K. Burley)