Proteomics & Mass Spectrometry

Core Resources
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Core Resources

Proteomics & Mass Spectrometry Guidelines

  • Starting a Project
    If your samples are for routine analysis (protein identification of gel or solution samples, HPLC purification, MALDI analysis for determination of protein molecular weight) and you know what services you need, please set up your ICBR account and submit a request to Proteomics and Mass Spectrometry. If your samples are not for routine analysis or you are not sure what the best approach is, please email ICBR-Proteomics@ad.ufl.edu and schedule a consultation or a project meeting.
  • How do I prepare a gel or solution samples?
    For direct infusion samples, MALDI-TOF analysis or 2D-GEL experiments, avoid or minimize salts in general, and desalting is recommended. Ammonium salts may be used at concentrations of 5- 25 mM.
    Avoid or minimize detergents, especially polyethylene glycols (Triton) and CHAPS. If detergent is needed use octyl β-D-glucopyranoside. Avoid viscous compounds (DMSO, glycerol). For gel samples, you can cut out the spot or band and place it in a 1.5 mL standard micro test tube. No need to add any solution. Avoid keratin contamination by using powder-free nitrile gloves when handling samples. Solution samples should not contain high concentrated detergents because they interfere with trypsin digestion. If you are not certain, please email your buffer composition to ICBR-Proteomics@ad.ufl.edu and we will review it.
  • How do I clean up a protein sample?
    Please always check with the core to determine if you need to clean your samples. If you do need to clean your protein sample, you can do either an acetone precipitation or chloroform-methanol precipitation.
  • How do I download Scaffold, ProteinPilot or Proteome Discoverer?
    If your result is in Scaffold format, you can download a free viewer (Scaffold Q+/Q+S) to view the data. For ProteinPilot, if you have Windows Professional, you can download a temporary trial version (30 days) of ProteinPilot to view the data.For Proteome Discoverer, you can download a trial. After 60 days the trial version will only act as a viewer.
  • What is the turn-around time?
    For routine analysis such as protein identification, results take no more than two weeks. For iTRAQ or label-free shot-gun sequencing, results normally take three to six weeks. Phosphorylation identification results take two to three weeks. Turn-around time depends on instrument availability and how many samples are in the queue. We provide weekly updates on your sample status.
  • How do I deliver samples to Proteomics and Mass Spectrometry Core?

    Drop off samples without an appointment between 9 a.m. and 5 p.m. To ship, send via FedEx Overnight using dry-ice boxes. We cannot accept weekend delivery, so please ship early in the week.

    • Mail to:
      ICBR Proteomics
      2033 Mowry Road
      CGRC building, ICBR Rm 169
      Gainesville, FL 32610ATTN: Jin Koh, PhDTel: +1-352-273-8060

Protocols

Protein Extraction

2D-DIGE

Isobaric labeling Quantification (iTRAQ/TMT/iodoTMT)

SILAC

Label-Free Shotgun Proteomics

MRM/SRM and PRM

PTM Sample Preparation and Analysis

Protein N-terminal Labeling and Analysis

Deliverables & Data Visualization

Deliverables

  • -iTRAQ: Raw files and ProteinPilot 5.0 or Scaffold with Mascot 2.6 output (please choose one)
  • -TMT: Raw files and Proteome Discoverer 2.2 output
  • -Label-Free Proteomics: Raw files and Scaffold Q+S with Mascot 2.6 or Proteome Discoverer 2.2 output
  • -identification: Raw files and Scaffold Q+S with Mascot 2.6 (*.dat)

Scaffold Viewer

http://www.proteomesoftware.com/products/qplus-s/

ProteinPilot Viewer

https://sciex.com/products/software/proteinpilot-software

Proteome Discoverer Viewer

https://thermo.flexnetoperations.com/

Mascot Public Search

http://www.matrixscience.com/cgi/search_form.pl?FORMVER=2&SEARCH=MIS

Policies

•At the conclusion of a project, all extra samples will be returned to the clients only if such request was made at the time of sample submission. The Proteomics facility will not store samples long term for clients.

•At the conclusion of a project, data given to clients will include all raw data files along with processed data files and report. It will be the sole responsibility of the client to keep and store these files in a safe place.

•If on future dates the data files have to be located, processed or re-analyzed by the Proteomics facility, clients will need to supply as much information regarding the samples and files as possible. This service will be charged at a minimal fee to cover the time and effort of Proteomics core personnel. It is the sole responsibility of the clients to keep the data files and Proteomics facility is not obligated to keep these files.

Acknowledging Proteomics & Mass Spectrometry

Authorship credit should be given if members of the UF ICBR Proteomics & Mass Spectrometry staff have provided services or expertise that is critical to a manuscript. Although other circumstances may warrant authorship, authors are those that meet the items below:

  • Agrees to be accountable for accuracy and/or integrity of the work and expertise. (In all our work the person executing the work takes the fullest degree of responsibility for accuracy and integrity.)
  • Provides substantial contributions to the experimental design, acquisition, and analysis/interpretation of the work.
  • Provides input on drafting of the work (often figures) and approves of the work to be submitted/published. (If a member of the facility has made substantial contributions it is expected that s/he will be given the opportunity to review drafts of manuscripts for accuracy/fidelity and should indicate agreement before a draft is moved forward to publication.)

If a member of UF ICBR Proteomics & Mass Spectrometry has contributed work to a manuscript, but does not meet the criteria for authorship, the corresponding author should acknowledge the UF ICBR Proteomics & Mass Spectrometry staff member who provided the data and their contributions should be specified.

These authorship expectations were developed in accordance with several other documents that outline criteria for authorship:

https://oir.nih.gov/sites/default/files/uploads/sourcebook/documents/ethical_conduct/guidelines-conduct_research.pdf

http://www.icmje.org/recommendations/browse/roles-and-responsibilities/defining-the-role-of-authors-and-contributors.html

Proposal Support

The Proteomics & Mass Spectrometry core provides a variety of services related to protein fractionation, qualitative and quantitative mass spectrometry analysis, de novo protein sequencing and protein database searching. Protein sequencing and identification are routine procedures in the lab. The capabilities in quantitative proteomics include iTRAQ (Isobaric Tags for Relative and Absolute Quantitation), TMT (Tandem Mass Tags), SILAC (Stable Isotope Labeling of Amino acids in Cell cultures), and MRM (Multiple Reaction Monitoring)-based absolute protein and metabolite quantification. Along with our capacities of separating thousands of proteins and characterizing differential protein expression, we have a suite of state-of-the-art mass spectrometers available for biomedical and advanced technology research, including a MALDI-TOF/TOF (4700 Proteomics Analyzer, AB), a quadrupole time-of-flight instrument (QSTAR Elite, AB), a quadrupole-linear ion-trap (4000 QTRAP, AB), a LTQ XL Orbitrap system, a Q-Exactive Plus Orbitrap, a TSQ Altis, and an Orbitrap Fusion (Thermo Scientific). These instruments are mainly used for accurate molecular weight determination, protein identification, post-translational modification, expression analysis (using in vitro isotope labeling (iTRAQ and TMT), SILAC in vivo labeling, and label free). Our facility is also set up to provide mass spectrometry-based N-terminal protein sequencing, sample preparation, and fractionation using HPLC. To ensure success and maximize productivity, the facility offers education, consultation, data processing and reporting, and support of grant application.

The core also provides a variety of services related to Biomarker Discovery, such as protein abundance analysis using gel-based 2 -D Fluorescence Difference Gel Electrophoresis (2-D DIGE); protein separation using 1-D or regular 2-D gel electrophoresis and Western blotting; 2-D gel-based phosphoprotein and glycoprotein profiling; Luminex biomarker discovery. For these services, we have instruments such as Ettan IPGphor, Ettan DaltSix, Typhoon Trio+ phosphor scanner, Investigator ProPic automatic spot picker, and FlexMap3D. In addition, we have image analysis software, such as GE DeCyder and Non-Linear Dynamics SameSpot Progenesis with Stats. Furthermore, the core provides protein enrichment and abundant protein depletion from biological fluids such as plasma, CSF, and urine using different techniques such as combinatory peptide (ligand) library (ProteoMiner), and assorted immunodepletion. The core also provides liquid phase preparative IEF to fractionate protein or peptide mixture using an Agilent 3100 Offgel fractionator.