Proteomics and Metabolomics, a part of systems biology, is fundamental for a full
understanding of biological systems. Mass spectrometry based technologies have been
indispensable to the development of molecular medicine. Clinical research can also
benefit from proteomics and metabolomics by both the identification of new drug targets
and the development of new diagnostic markers.
The Georgia Cancer Center's Proteomics and Metabolomics core service uses mass spectrometry
as the key technology for qualitative and quantitative protein characterization and
advanced methods for profiling and quantitation of small molecules. Our principal
approach for protein analysis is 'bottom-up' proteomics, where all proteins are proteolytically
digested, producing peptide surrogates (signature peptides) of the original proteins.
With highly specialized technological capabilities, proficiency and expertise of personnel
in the field of mass spectrometry, the facility is well-prepared and positioned to
serve as a regional, national, and international resource and to foster collaboration
in the fast developing field of proteomics and metabolomics.
Please, submit your service request via iLAB.
- Sample extraction and purification for metabolomics
- Metabolome profiling
- LC/MS analysis of the different sample sets (i.e., disease vs. matched controls)
- LC/MS differential analysis of the LC/MS analyses
- Provide LC/MS output (m/z and RT) highlighting the ions that differed the most in
intensity between data sets
- Provide tentative identification based on accurate mass and molecules available in
the METLIN database
- Provide firmer identification based on comparative MS/MS and high accuracy analysis
of 'unknown' with a standard provided by the client
- Protein extraction from cell lines, tissues and biofluids
- Trypsin digestion
- Protein identification
- Detection and characterization of post-translational modifications of proteins, structural
characterization of modified proteins, lipids and DNA in disease, e.g., the identification
and quantification of oxidative damage to proteins, lipids and DNA
- Screening for genetic mutations in proteins
Meet The Team
Thermo Fisher Scientific LTQ Orbitrap Velos Pro Hybrid FT MS
- Thermo Scientific Orbitrap Velos Pro
- Thermo Scientific Dionex Ultimate 3000 RSLCnano
The Orbitrap LC-MS systems deliver a maximum resolution (FWHM) of 1,000,000 at m/z
200 and a sub-1 ppm mass accuracy in a single compact and easy-to-use instrument.
These high-resolution accurate-mass systems detect a wide range of compounds including
small molecules during both targeted and untargeted analyses, without losing selectivity
Key benefits of using this system:
- Nanometer-range accuracy electrodes.
- Mass-to-charge measurements delivered as a function of oscillation frequency.
- Due to the superior resolving capabilities of Orbitrap technology, accurate mass assignment
is easily achieved, with sub-1-ppm mass accuracy typical of injected analytes.
Agilent Technologies Q-TOF
- Agilent 6520 Accurate-Mass Quadrupole Time-of-Flight MS
- Agilent 1200 Series Binary LC System
Agilent 6520 Accurate-Mass Q-TOF LC/MS
Because Orbitrap technology provides quantitative and qualitative (quan/qual) HRAM
analytical capability in a single platform and often in a single run, it has been
increasingly applied towards a range of challenging applications requiring high selectivity
6520 Accurate-Mass Q-TOF LC/MS, equipped with 1200 Series Binary LC system, provides
mass resolution power up to 40,000 with sub-1-ppm MS and 2-ppm MS/MS.
This system provides:
- Data dependent LC/MS/MS proteomic analyses.
- Metabolomic profiling and biomarker discovery/validation.
- Increased specificity and sample definition by combining accurate mass measurement
with quadrupole time-of-flight mass spectrometry.
Agilent Technologies QQQ
- Triple Quadrupole MS systems
- Agilent 6470B Triple Quadrupole LC/MS system
- Agilent 1260 Infinity II Binary Pump
Agilent 6470B LC/TQ
The Agilent 6470B LC/TQ, the latest most advanced version of triple quadrupole systems,
provides the versatility and robustness that is needed for many applications. With
its proven performance as demonstrated in a number of publications, the 6470B LC/TQ
comes with multiple technological advances like the Agilent Jet Stream ion source,
curved geometry collision cell, and ±20 kV high-energy dynode.
It provides maximized ion formation; reaches detection limits up to the sub-femtogram
level with Agilent Jet Stream (AJS) source. Wide mass range allows the analysis of
ions of various classes and sizes (small molecules to multiple charged peptides).
The 6470B Triple Quadrupole LC/MS system analyzes complex samples, screens, confirms
and quantifies with triggered MRM (tMRM), which combines fast and sensitive MRM quantitation
with the generation of a product ion spectrum for library searching and compound screening
Sample Preparation Protocols
Sample Preparation Protocol for cells (adherent and suspension, 10 cm2 plates)
(Note: A sample consisting of ≈ 5 µL of cell pellet, which would require approximately
2.0 x108 cells, would be sufficient to yield successful metabolomics analyses.)
- For adherent cells, wash plate with 10 mL of medium. Remove medium completely.
- Incubate for 2 hours in a fresh 10 mL aliquot of medium. Decant excess medium. Aspirate
off residual medium.
- Immediately add 5 mL of Chloroform/Methanol/Water: (1:3:1 ratio; at -80°C), and transfer
to -80°C incubator. During the transport of the cell mixture to the -80°C incubator,
the contents of cell culture must be submerged, at all times, in a Dry Ice/Ethanol
bath, held at -80°C.
- Incubate mixture for 15 minutes
- After 15 minutes of incubation, scrap plate with a cell scraper (while still submerged
in Dry Ice/Ethanol bath at -80°C).
- Transfer cell lysate to 15 mL conical tube that is submerged into a Dry Ice/Ethanol
bath at -80°C.
- Centrifuge at full speed for 5 minutes in cold room.
- Transfer supernatant to a fresh 15 mL conical tube that is submerged in Dry Ice/Ethanol
bath at -80°C.
- To the pellet in the 15 mL conical tubes, add a fresh aliquot of 500 μL of the extraction
solvent (Chloroform/Methanol/Water: (1:3:1 ratio; at -80°C)). Re-suspend thoroughly;
may require sonication.
- Transfer mixture to 1.5 mL Eppendorf® tube submerged in Dry Ice/Ethanol bath at -80°C.
- Spin in micro centrifuge at full speed for 5 minutes in cold room.
- Transfer supernatant to the 15 mL conical tube that is submerged in Dry Ice/Ethanol
bath at -80°C (of step 8).
- Add 500 μL of the extraction solvent (at -80°C) to the pellet in the 1.5 mL Eppendorf®
- Spin in micro centrifuge at full speed for 5 minutes in cold room
- Transfer supernatant to the 15 mL conical tubes (in Dry Ice/Ethanol bath at -80°C)
(of step 8).
- Evaporate pooled extracts to dryness using a speedVac or a lyophilizer.
- Submit dried pellet in 1.5 ml Eppendorf® tube (can be stored at -80°c).
Protocols for trypsin digestion
A. In-solution digestion protocol
Reference: Kinter, M., and Sherman, N. E. (2005) The Preparation of Protein Digests
for Mass Spectrometric Sequencing Experiments. in Protein Sequencing and Identification
Using Tandem Mass Spectrometry. pp 147-165
- 1M NH4HCO3 (ABC): 79 mg/mL ultra pure water (fresh, keep on ice)
- Reducing reagent – 200 mM dithiotreitol (DTT)/100 mM ABC: dissolve 31 mg DTT in 900-µL
of ultra pure water and add 100 µL of 1M ABC.
- Urea solution – 8M urea/100mM ABC/5mM DTT. Place 480 mg urea in tube, add 100 µL of
1M ABC, add 25 µL of 200 mM DTT, and adjust the volume to 1 mL with ultra pure water.
- Alkylating agent – 200 mM iodoacetamide (IAA)/100 mM ABC: dissolve 37 mg IAA in 900
µL of MS water and add 100 µL of 1 M ABC.
- Buffer for dilution: 50 mM ABC/2 mM CaCl2.
- Trypsin solution – 0.2 µg/µL. Prepare just before use. First, 50 mM ABC/2 mM CaCl2,
keep on ice. Add 100 µL of ice-cold 50 mM ABC/2 mM CaCl2 to 20 µg of sequencing-grade
modified trypsin (Promega). Dissolve trypsin by drawing the solution into and out
of the pipette. Keep this solution in ice until use.
The protein sample (we used pellet after Amersham clean-up) is evaporated and re-suspended
in urea solution (i.e. with DTT; solution #3 above) up to the protein concentration
~ 5 µg/µL. Mix carefully by drawing the sample into and out of the pipette.
- Reduction: 1-2 hour at room temperature or 1 hr at 30ºC in a mixer. Adjust to room
temperature and spin down.
- Alkylation: Add alkylating reagent (200mM IAA) up to 15 mM to the reaction mixture.
Incubate 45 minutes at room temperature in the dark.
- Add the same amount of the reducing agent to consume any un-reacted IAA. Incubate
~ 20 minutes (Example: add 1.5 µL of 200 mM DTT).
- Reduce the urea concentration up to 1 M by diluting the reaction mixture with 50 mM
ABC/2 mM CaCl2 (Example: add 140 µL of 50 mM ABC/CaCl2 to 20 µL of starting sample
- Add trypsin solution to the reaction mixture at 1:30 – 1:40 ratio. Vortex gently,
put for digestion overnight (16 – 18 hr) at 37 ºC (Example: if a total protein amount
– 100 µg, add 12.5 µL of 0.2 µg/µL trypsin solution to achieve 1:40 ratio).
- Next Day: Stop reaction by adding concentrated (10%) TFA or FA and adjust pH to ~
- Add also acetonitrile (ACN) up to 2% if you plan to desalt the digest by micro, Macro
trap desalting cartridges (see Michrom guide to Trap Cartridge care and use). Test
the pH by placing the 1-L aliquots of the samples onto an appropriate pH paper.
- Desalted digest can be analyzed by 2D LC/MS/MS. For 1D LC/MS – (if you have peptide
Trap in-line to wash out reagents) you can use un-desalted digest. Keep unused digest
B. In-Gel Digestion Protocol
University of California, San Francisco Mass Spectrometry Facility
C. Protocol for immunoprecipitation
Teng, Y., Ngoka, L., Mei, Y., Lesoon, L., and Cowell, J. K. (2012) HSP90 and HSP70 Proteins Are Essential for Stabilization and Activation of WASF3 Metastasis-promoting
Protein. The Journal of biological chemistry 287, 10051-10059.
Teng Y, Ngoka L, Cowell JK: Promotion of invasion by mutant RAS is dependent on activation of the WASF3 metastasis
promoter gene. Genes Chromosomes Cancer 2017, 56:493-500.
Dytfeld D, Rosebeck S, Kandarpa M, Mayampurath A, Mellacheruvu D, Alonge MM, Ngoka
L, Jasielec J, Richardson PG, Volchenboum S, Nesvizhskii AI, Sreekumar A, Jakubowiak
AJ: Proteomic profiling of naive multiple myeloma patient plasma cells identifies pathways
associated with favourable response to bortezomib-based treatment regimens. Br J Haematol 2015, 170:66-79.
Teng Y, Ghoshal P, Ngoka L, Mei Y, Cowell JK: Critical role of the WASF3 gene in JAK2/STAT3 regulation of cancer cell motility. Carcinogenesis 2013, 34:1994-9.
Wang X, Choi JH, Ding J, Yang L, Ngoka LC, Lee EJ, Zha Y, Mao L, Jin B, Ren M, Cowell
J, Huang S, Shi H, Cui H, Ding HF: HOXC9 directly regulates distinct sets of genes to coordinate diverse cellular processes
during neuronal differentiation. BMC Genomics 2013, 14:830.
Teng Y, Ngoka L, Mei Y, Lesoon L, Cowell JK: HSP90 and HSP70 proteins are essential for stabilization and activation of WASF3 metastasis-promoting
protein. J Biol Chem 2012, 287:10051-9.
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