Question 1

Should I use TIMS-TOF or Orbitrap Eclipse?

Accepted Answer

Both instruments are state-of-the-art instruments in bottom-up proteomics.  The major difference between the two instruments is the mass resolution of the MS2 spectra. With a higher mass resolution, Orbitrap Eclipse supports TMT quantitation up to 18-plex (i.e., 18 channels).  In terms of Protein IDs, the proteins identified by the two instruments may be different as the fragmentation in TIMS-TOF is induced by CID and that in Orbitrap Eclipse is by HCD. Yet, in general, the number of proteins identified by the two instruments is similar under standard operating conditions.

Question 2

What are the differences between Ion Trap and Orbitrap detection mode in Orbitrap Eclipse?

Accepted Answer

Orbitrap Eclipse can be operated in two modes, Ion Trap detection mode and Orbitrap detection mode. Ion Trap provides a high scan rate so that more MS2 spectra can be acquired, resulting in an increased number of protein IDs. Orbitrap provides a high mass accuracy and a high resolution which is essential for separating the TMT reporter ions. Thus, it is recommended to use the Ion Trap detection mode to maximize the number of protein IDs. But for TMT quantitation, Orbitrap detection mode must be used.

Question 3

What is FAIMS in Orbitrap Eclipse?

Accepted Answer

The High Field Asymmetric Waveform Ion Mobility Spectroscopy (FAIMS) technology reduces chemical noise and matrix interferences for improved robustness and sensitivity. The separation of ions is based on a combination of factors such as charge state, shape, conformation, and gas-phase ion size. Please contact BioCRF staff or refer to the Thermo Scientific website here for more details.  BioCRF’s Orbitrap Eclipse is coupled with the Thermo Scientific™ FAIMS Pro™ interface to increase ion selectivity for identifying more proteins. By default, the proteomic analysis using Orbitrap Eclipse is always conducted with the FAIMS Pro™ interface. Please contact BioCRF staff if the samples have to be analyzed without the FAIMS Pro™ interface.

Question 4

What samples can be submitted?

Accepted Answer

We routinely accept (1) cell lysates and (2) cell pellets (for mammalian cell lines only). Please submit the samples in 1.5 mL centrifuge tubes.  For cell lysates, viscous samples will NOT be accepted. Perform DNA shearing with a Microtip Sonicator or add Benzonase to cleave the DNA if necessary. For optimal results, please submit at least 10 micrograms of protein.  For cell pellets, cell pellet submission is applicable for mammalian cell lines only. It should be noted that the number of protein IDs obtained from cell pellet is normally less than that of cell lysate since a separated cell lysis step is omitted. However, when the number of cells is limited, samples submitted in cell pellet form may result in an increased number of protein IDs since fewer processing steps are involved and may result in less sample loss. Yet, BioCRF will not guarantee that the number of protein IDs must increase when cell pellets are submitted.  For protein extracts, users should ensure that the proteins are properly extracted. Only clear, transparent protein solutions will be accepted. If necessary, please perform multiple centrifuge cycles or even ultracentrifuge to remove any precipitate. For optimal results, please submit at least 10 micrograms of protein.  For gel bands, they are no longer accepted. Users have to perform in-gel digestion by themselves and submit the resultant peptides for analysis. Please refer to the in-gel digestion protocol here.  For ready-to-inject samples, they can be submitted. However, to safeguard the sample quality, an extra C-18 desalting step will be performed by BioCRF.  For other samples, their submission is subjected to case-by-case approval. Please contact BioCRF staff before sample submission.  Moreover, in case there is any blockage or malfunction of the LC system due to poor sample quality, BioCRF reserves the right to charge the users for the consumable cost, including but not limited to the injector, solvent pumps, tubings, LC columns, and emitter.

Question 5

How to submit samples?

Accepted Answer

For HKUST users, please send the sample submission form to BioCRF staff via email. Please submit the samples (together with the sample submission form) to Rm 6127A during office hours. Otherwise, please submit the samples to the 4°C/ -20 °C refrigerator right next to the front door of Room 6127 and contact our staff via email after sample submission.  For external users, please consult BioCRF staff before sample submission.

Question 6

How to fill in the sample submission form?

Accepted Answer

Please refer to the form filling guidelines here.

Question 7

Is the TMT labeling service available in BioCRF?

Accepted Answer

BioCRF does not provide the TMT labeling service.  Users can submit their protein samples (e.g., cell lysates) to BioCRF for digestion. The digested samples, which are ready for TMT labeling, will be returned to the users and users have to perform TMT labeling by themselves. After TMT labeling, users should combine equal portions of samples from each channel and return this solution mixture to BioCRF. BioCRF will then perform desalting (this desalting step must be performed by BioCRF staff to ensure the quality of the final product) and MS analysis. Since a desalting step has to be performed by BioCRF, an additional material charge will be incurred.  Please refer to the instructions from Thermo Scientific (link) or consult BioCRF staff for detailed TMT labeling procedures.

Question 8

Is it possible to aliquot the TMT labels and store them for the next use?

Accepted Answer

TMT labels are NHS-esters that are highly sensitive to water/moisture.  According to the manufacturer, anhydrous acetonitrile is the recommended solvent to dissolve the TMT reagents. Stock solutions are stable for one week when stored at -20°C. For long-term storage of unused reagents, remove all solvent by drying and store with desiccants at -20°C.  Please discuss with BioCRF staff for more information on aliquoting/storing TMT labels. Yet, BioCRF will not be responsible for any consequence and does not guarantee the labeling efficiency of the aliquots/unused reagents.

Question 9

What are the differences between MS2 and MS3 TMT Quantitation in Orbitrap Eclipse?

Accepted Answer

MS2 and MS3 represent the MS order in which the TMT reporter ions are collected. Simply, more protein IDs can be obtained using MS2 quantitation while more accurate quantitation results can be achieved by MS3 quantitation.  Please refer to the application note (link) or consult BioCRF staff for further information on MS2 and MS3 quantitation.  The MS3 quantitation mode used in BioCRF’s Orbitrap Eclipse is real-time search synchronous precursor selection MS3 (RTS-SPS-MS3).

Question 10

Why is the number of protein IDs reduced after TMT labeling?

Accepted Answer

One of the major reasons is related to the difference in tandem MS settings. In TMT quantitation, the MS2 or MS3 ion injection time is increased to collect more TMT reporter ions for accurate quantification, sacrificing the scan speed and resulting in a decreased number of protein IDs. Other reasons include sample loss during TMT labeling/ desalting may also reduce the number of protein IDs. To improve the number of protein IDs, users may consider performing high pH reversed-phase peptide fractionation (reference) after combining the TMT-labeled peptides.

Question 11

How can I open the MS raw data?

Accepted Answer

The MS data from TIMS-TOF Pro is in .d format, which can be opened by the software DataAnalysis (Bruker). The MS data from Orbitrap Eclipse is in .raw format, which can be opened by the software QualBrowser (Thermo). Please consult BioCRF staff for the corresponding software if necessary.

Question 12

What are the database search parameters?

Accepted Answer

The database search of Orbitrap Eclipse data is performed using Proteome Discoverer and the tims-TOF Pro data is searched using PEAKS Xpro. Please find the database search parameters and the output here and consult BioCRF staff in case another set of parameters should be used.

Question 13

What can be obtained from the database search?

Accepted Answer

Please find the database search parameters and the output here. Depending on the searching mode (e.g., Protein ID or LFQ), the output may be different. For instance, information related to protein abundance cannot be obtained for the Protein ID search.

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