Mass Spectrometry Facility
Introduction
The Carnegie mass spectrometry facility adapts and develops proteomic tools to drive biological studies. The facility is in close collaboration with various groups at Carnegie and labs at Stanford biology department.
The facility includes one benchtop quadrupole Orbitrap mass spectrometer (Q-Exactive HF) coupled with nano-flow UHPLC liquid chromatography (Thermo Scientific Easy-nLC 1200 system). The facility is also equipped with AKTA purifier 10 and pure 25 M chromatography system.
The facility provides analysis for identification of both proteins and post-translation modifications, including phosphorylation, ubiquitination, methylation and O-GlcNAcylation. The facility is also providing cross-linking analysis for mapping protein structure of functional protein complexes. For quantification, the facility provides both MS1 based quantification (spectra count and label-free quantification-LFQ, SILAC-based N15 metabolic labeling) and MS2-based quantification (Parallel Reaction monitoring-PRM). Tools used by Carnegie mass spectrometry facility for analysis of large mass spectrometry proteomics data include Protein Prospector, MaxQuant, Perseus and Skyline. An internal Carnegie server is set up for deposition and processing of MS data.
For questions, please contact the proteomic director Shouling Xu, sxu@carnegiescience.edu, 650-739-4225.
Instruments
Resource Type:
Chromatograph
Make:
ThermoFisher
Features:
This nano-flow UHPLC provides a reliable, robust and easily serviceable system for peptide separations.
Specifications:
PRESSURE RANGE 0 to 1200 bar
0-1200 bar
MAX. FLOW RATE 2000nL/min.
MIN. FLOW RATE 20nL/min. (Recommended 100nL/min.)
Resource Type:
Spectrometer
Make:
ThermoFisher
Specifications
MASS RANGE 50 to 6,000 m/z (up to 8,000 m/z with BioPharma option)
DYNAMIC RANGE >5,000:1
MASS ACCURACY Internal: <1 ppm RMS; External: <3ppm RMS
POLARITY SWITCHING
One full cycle in < 1 sec (one full positive mode scan and one full negative mode scan at a resolution setting of 60,000)
SENSITIVITY
MS/MS: 100 fg reserpine on column S/N 150:1
SIM: 50 fg reserpine on column S/N 150:1
RESOLUTION Up to 240,000 (FWHM) at m/z 200
SCAN RATES Up to 18 Hz
Resource Type:
Chromatograph
Make:
General Electric
Model:
ÄKTA pure 25 M
Coupled with autosampler and fraction collector, for peptide off-line seperation, using either isocratic flow, size-exclusion chromatography, ion exchange chromatography, or Reverse phase high-pH HPLC.
Specifications:
Application Flexible and intuitive chromatography system for fast purification of proteins, peptides, and nucleic acids from microgram to gram levels of target product.
Application supported Affinity chromatography, size exclusion chromatography (SEC, also known as gel filtration), ion exchange chromatography, hydrophobic interaction chromatography, and reversed phase chromatography (RPC). For RPC runs use only Frac F9-R.
Flow rate 0.001 to 25 mL/min (normal range), 0.01 to 50 mL/min (column packing flow)
Operating pressure max. 20 MPa (2900 psi)
Operating pressure (sample pump) max. N/A
Tubing i.d. (flow path) 0.5 mm
Multiple wavelength detection Up to 3 wavelengths
UV wavelength 190 to 700 nm
Flow cell path length 2 mm (optional 0.5 mm and 10 mm)
pH monitoring 0 to 14 (optional)
Multiple sample injection Upgradeable to 5 samples
Autosampler injection No
Automatic buffer preparation No
Buffer selection Upgradeable to 14 inlets
pH stability, cleaning-in-place (CIP) 0 to 14
pH stability, operational 2 to 12
Multistep purification Yes, (optional)
Relative humidity 20% to 95% (noncondensing)
Viscosity 0.35 to 10.0 cP (system pump)
Resource Type:
Chromatograph
Make:
General Electric
Model:
ÄKTApurifier 10
With fraction collector, in cold-box, for protein seperation using size-exclusion chromatography or ion exchange chromatography.
Resource Type:
Bioreactor
Make:
Nicoyalife OpenSPR
Features:
This OpenSPR, a surface plasmon resonance instrument, from Nicoya, provides high quality, label-free interaction analysis for protein-protein, protein-DNA/RNA, protein-small molecules interactions.
Personnel
Shouling Xu
Director of Proteomics, PI
Cell mapping, structural analysis using cross-linking mass spectrometry, nutrient sensing
Office: 650-739-4225
Email: sxu@carnegeiscience.edu
Su Hyun Hong
Research Associate
Email: shong@carnegiescience.edu
Danbi Byun
Research Assistant
Study area: nutrient sensing ; structural analysis using cross-linking mass spectrometry;
Email:dbyun@carnegiescience.edu
Rick Kim
Joint Research Associate, Grossman Lab and Xu Lab
Area: Study of lysine methylation in Clamy
Senior Users
Weimin Ni
Postdoctoral Scholar, Grossman Lab
LC-MS analysis of regulatory proteins and protein complexes
Email: wni@carnegiescience.edu
Expertise:
Calibration and Maintenance of HPLC and MS instrument; Data analysis using Protein Prospector and Maxquant; PRM set up and data analysis using Skyline; Quantitative Mass Spectrometry analysis using Metabolic labeling and LFQ; Post-translational modifications analysis, HPLC (AKTA) –Reverse HPLC, Ion Exchange and Size Exclusion Chromatography, Sample prep from various tissues.
Chuan-Chih Hsu
Postdoctoral Associate, Wang Lab
Study of phosphorylation events in plants under environmental stresses
Expertise: Skilled user of HPLC and MS instrument; Data analysis using Protein Prospector and Maxquant; Quantitative Mass Spectrometry analysis using LFQ; Post-translational modifications analysis; Sample prep method development.
Selected Publications
Mair A, Xu SL, Branon TC, Ting AY, Bergmann DC. Proximity labeling of protein complexes and cell-type specific organellar proteomes in Arabidopsis enabled by TurboID. 2019. BioRxiv (under review of eLife). doi: https://doi.org/10.1101/629675.
Kim TW, Park CH, Hsu CC, Zhu JY, Hsiao Y, Branon TC, Xu SL, Ting AY, Wang ZY. Application of TurboID-mediated proximity labeling for mapping a GSK3 kinase signaling network in Arabidopsis. 2019. BioRxiv (under review of eLife).
Garcia VJ, Xu SL, Raksha R, Wang W, Elliott L, Fensenko M, Altmann M, Falter-Braun, P, Moore I, Assadd FF, Wang ZY. Proteomic Studies of the Arabidopsis TRAPP complexes reveal conserved organization and a novel plant-specific component with a role in plant development. 2019. (under review of Plant Cell).
Kim EJ, Lee SH, Park CH, Kim SH, Hsu CC, Xu S, Wang Z, Kim SK, Kim TW. Plant U-Box 40 Mediates Degradation of the Brassinosteroid-Responsive Transcription Factor BZR1 in Arabidopsis Roots. Plant CELL. 2019.
Xu SL, Chalkley JR, Maynard JC, Wang W, Ni W, Jiang XY, Shin K, Cheng L, Savage D, Huhmer AFR, Burlingame AL, and Wang ZY. 2017. Proteomic analysis reveals O-GlcNAc modification on proteins with key regulatory functions in Arabidopsis. PNAS, 114, E1536–E1543. [ PubMed]
Ni W*, Xu SL*, Grandio EG, Chalkley RJ, Huhmer AF, Burlingame AL , Wang ZY and Quail PH . 2017. PPKs mediate direct signal transfer from phytochrome photoreceptors to transcription factor PIF3. Nat Commun , 8, 15236. [PubMed] Highlighted in F1000.
Xu SL, Medzihradszky KF, Wang ZY, Burlingame AL, Chalkley RJ. 2016.N-Glycopeptide profiling in Arabidopsis inflorescence.MCP. 15(6), 2048-2054. [ PubMed].
Ni W *, Xu SL *, Tepperman JM, Stanley DJ, Maltby DA , Gross JD, Burlingame AL , Wang ZY and Quail PH . 2014. A mutually assured destruction mechanism attenuates light signaling in Arabidopsis. Science , 344, 1160-64. [ PubMed] Highlighted by Hines (2014), Emerging from the shade into the light, Sci Signal. 7 (329): ec161, 2014; Highlighted in F1000; Spotlighted by Zhu & Huq, 2014, Suicidal codegradation of the Phytochrome Interacting Factor 3 and phytochrome B in response to light. Mol. Plant, doi:10.1093/mp/ssu108.
Kim EJ, Youn JH, Park CH, Kim TW, Guan S, Xu S, Burlingame AL, Kim YP, Kim SK, Wang ZY, Kim TW. 2016. Oligomerization between BSU1 Family Members Potentiates Brassinosteroid Signaling in Arabidopsis. Mol Plant , 9(1): 178-181. [ PubMed]
Xu P*, Xu SL*, Li ZJ, Tang W, Burlingame AL, Wang ZY. 2014. A brassinosteroid-signaling kinase interacts with multiple receptor-like kinases in Arabidopsis. Mol Plant , 7(2):441-4. [ PubMed]
Ni W *, Xu SL *, Chalkley RJ , Pham TN , Guan S , Maltby DA , Burlingame AL , Wang ZY and Quail PH . 2013. Multisite light-induced phosphorylation of the transcription factor PIF3 is necessary for both its rapid degradation and concomitant negative feedback-modulation of photoreceptor phyB levels in Arabidopsis. Plant Cell, 25: 2679-98. [ PubMed]