FAQ

 

(1) How do I submit and ship my samples to the facility?
(2) How do I get my results?

Data result reports are sent by e-mail as PDF, Excel, Word or PowerPoint files.

(3) What is the data return time for protein identification, MW determination, protein modification characterization, and quantitative proteomics?

The facility provides routine analysis services on a first come, first serve basis.  Data return time for routine analysis services is usually 5-8 business days after we receive samples.  Data return time is usually 7-10 business days for protein modification characterization services.  Data return time is usually 2-3 weeks for quantitative proteomics services.

(4) What services are availble for 2D gel analysis?

The facility will provide 2D gel equipment and software support for users’ conducting 2D DIGE experiments or other 2D gel approach followed by SameSpots or DeCyder software analysis for global quantitative proteomics analysis.  Facility doesn’t offer 2D DIGE or and/or by 2D gel-based quantitative analysis for global quantitative proteomics anymore. The facility will continue to provide services for running both IEF and SDS-PAGE. We will stain the gels (colloidal coomassie blue, Sypro Ruby stain), scan image in Typhoon 9400 laser scanner and send those image files to the investigator for general protein separation purpose or subsequent associated western blotting analysis. The facility will allow the users to have access to image analysis software for annotating the image and image data analysis. The investigator will determine the excision of interesting spots and submit request for subsequent LC-MS analysis. The facility will assist investigators in interpreting results and suggesting further experiments, but detailed interpretation and experiments beyond usual protein gels will be outside the ordinary business of the facility.
(5) Who is responsible for sample preparation for 2D gels?

Sample preparation and sample QA/QC (e.g., verification that a reasonable amount of protein is present) are done by the investigator.  Please follow our 2D gel sample preparation guideline.   In addition, the facility can provide consultation on sample preparation for 2D gel analysis.

(6) What do I need to do for 1D gel bands?

You can submit 1D gel bands for 1D gel-based protein IDs if you have relatively less complex samples in a separated fraction or from immunoprecipitated proteins.  Make sure you cut out only the gel band containing proteins (stained portion).  For the first time user, please bring your gel to the facility and we will help you excise the gel bands/spots.

(7) What stain method should I use for my 1D gel band and 2D gel spots?

It depends on what detection level you need.  Optimal estimated detection limits are: 50-100 ng for standard coomassie; 10 ng for colloidal coomassie; 1 ng for fluorescent dyes; and 0.5-1 ng for silver stain.  It should be noted that silver stain is one of the most sensitive stains, but because it is less compatible with the subsequent mass spectrometry analysis, we do not recommend using silver staining for any samples that will subsequently be submitted for MS analysis.  Please see our sample preparation protocols.

(8) How much sample do I need and why do I have to submit 1 mg of protein for 2D gel analysis?

That depends on many factors.  Obtaining sufficient protein from most sources typically requires extracting a few milligrams of tissue (wet weight) to get a few hundred micrograms of protein.  Very low abundance proteins (e.g., only a few copies per cell) may not be detectable at all by ordinary staining methods, no matter how much tissue you extract.  Other factors include sample complexity, size of 2D gel used for separation, the pI range targeted for separation and the staining method used for analysis.  Generally speaking, 200-300 micrograms of proteins will be sufficient for one 2D gel analysis.  Usually, 2D gels run in duplicate are required for evaluating reproducibility.  We recommend submitting enough protein for at least two runs (in case of problems with the first run).

(9) What is the detection limit for protein identification on 1D gel bands and 2D gel spot samples?

The sensitivity for protein identification by the Orbitrap Fusion or Orbitrap Elite (nanoLC-MS/MS) is around 1-10 fmol for most standard protein digests.  For 2D gel spots, if you can see it with colloidal Coomassie Blue, or with Sypro Ruby, even for low stained spots, there is a good chance for successful identification by nanoLC-MS/MS analysis.  For 1D gel bands, however, it will depend on the protein complexity on the gel band.  For SDS-gel samples, if a band contains possible multiple proteins or requires high sequence coverage (for identification of modifications or peptide mapping analysis), nanoLC-MS/MS analysis is required.

(10) What should I pay attention to for in-gel sample handling and for preparation for subsequent MS analysis?

The key point is to keep sample handling as clean as possible.  That is simply because contamination with other proteins could prevent identification of your interesting proteins, which might be present in relatively low abundance.  We request that you submit a blank control to be run in parallel with your other samples, as a background measurement.  The most common contaminants are human keratins and albumins.  The keratin comes from dust, skin, hair and fingerprints.  Even a small hair contains overwhelming amounts of keratin, which could be much more abundant than the proteins of interest in your sample. 

Pay particular attention to the following:   

  • Always wear powderless gloves, rinsed with water and ethanol or methanol prior to use.
  • Use freshly-made, high purity reagents and water to make buffer and gels.
  • Use clean dishes for gel casting as well as staining.
  • Use siliconized polypropylene tubes as well as low-retention tips to minimize protein loss by adsorption to tube walls. Do not use glass tubes.
  • Free acrylamide will react with the amino groups of proteins during polyacrylamide gel electrophoresis. Thus, always cast the separation gel and allow overnight polymerization prior to use.
  • Excise gel bands on extremely clean surfaces using new razor blades or scalpels.  Ideally, this should be done in a laminar flow hood (tissue culture type) to minimize the possibility of any dust, hair, flakes of skin, or other forms of debris contaminating the samples.

Please see our sample preparation protocols.

(11) What is the impact of the solvent and buffer used in my samples on the success of direct MS analysis?

Use of the appropriate solvents is very important for the subsequent mass spectrometry analysis.  Volatile solvents such as methanol and acetonitrile, are ideal solvents for direct mass spectrometry.  Avoid using DMSO, DMF, THF and chaotropes.  Always use spectroscopic grade solvent to prepare the sample.  Salts and buffers are, in general, detrimental to MS analysis.  Salts often form adduct peaks which compete with the molecular ion peaks and broaden the overall signal (especially for protein analysis).  ESI-MS is very sensitive to salts and buffers resulting in signal suppression.  Less than 1 mM salts and buffers are recommended.  Volatile salts such as ammonium acetate, ammonium formate, and ammonium biocarbonate, usually do not greatly affect the signal below 20 mM.  However, sodium and potassium can be a real problem even less than 1 mM.

(12) What services does the MS core facility provide for LC/MS/MS analysis?

The facility can provide either 1D LC-MS/MS or 2D LC-MS/MS analysis.  For relatively less complex samples (gel bands or in-solution digestion of fractions) with a certain dynamic range of analytes in complexity samples, the facility can provide direct online reverse phase LC (RPLC) separation with MS and MS/MS detection, which is operated in data dependent acquisition (DDA) mode with automatic dynamic exclusion activated for protein/peptide identification, quantitation, post-translational modification (PTM) characterization and small molecule quantitation and identification.  For more complexity proteome samples, 2D LC with an additional first dimensional LC in either offline or online fractionation coupled with RPLC-MS/MS analysis for each of the fractions is often required.  The most common, first dimensional LC used offline in the facility is high pH reverse phase LC for complexity peptide separation, which is based on UV absorbance at 214 nm and with a multiple fraction concatenation strategy for pooling fractions.  Quantitation analysis and PTM characterization are offered on a case-by-case basis, as the outcome of these analyses are often dependent on the inherent nature of submitted samples (analytes) and some may require analysis method development.  On-line LC/MS/MS analysis consists of both analytical scale LC/MS/MS equipped with a standard ion source and nanoLC/MS/MS equipped with a nano ion source.  Suitable samples for LC/MS/MS analysis include both gel-based samples and solution-based complex samples.  The LC/MS/MS approach can also be used for obtaining amino acid sequence information via de novo sequencing for proteins when there is no database available or there are some unknown modifications.  The facility can assist investigators in interpreting results and further experimental design.

(13) What samples require LC/MS/MS analysis?

Compared to infusion analysis, LC provides at least two key advantages, including separation of complex analytes and enrichment of analytes for enhancing the subsequent MS detection.  This is because multiple component samples will compete for protons in the ionization process causing ion suppression (weak signals).  Thus, pure samples always provide more satisfactory data.  In addition, the ESI/MS is a solution-based analyte concentration-dependent mass detector.  The components with the highest concentration will give the strongest signal and the components with low concentration will give weak signals.  Thus, LC/MS or LC/MS/MS analysis is required for any samples containing multiple analytes, particularly those with variable abundance.

(14) How do I prepare samples for LC/MS/MS analysis?

Samples with relatively high salt contamination and nonvolatile buffer can be analyized by coupling MS with up-front LC.  However, the sample should not contain hydrophobic contaminants or detergents such as PEG, Triton, SDS, CHAPS, NP-40 etc., since those polymers can not only suppress analyte ionization but also bind tightly to the RP column, and thus permanently damage the column separation resolution.  If your samples contain those hydrophobic contaminants, we will have to add a sample cleanup step prior to LC-MS analysis. In addition, samples should not contain more than 5% organic solvent such as ACN and methanol.  Reduce any organic solvent concentration by speed vacuum prior to LC analysis.  Please see our sample preparation protocols.

(15) What are the advantages and disadvantages between infusion ESI/MS analysis versus LC-ESI/MS analysis?

The advantages of LC/MS analysis includes reducing ion suppression and enriching target analyte concentration.  Thus, LC/MS usually provides better sensitivity than infusion analysis.  The main advantage of infusion analysis is that infusion can provide extended MS analysis time and allow you to optimize the MS condition for each of targeted ions with different ion modes.  In addition, the extended acquisition time offered by infusion allows one to sum the MS or MS/MS scans to improve the spectra quality.  However, infusion analysis usually consumes more sample and requires clean sample (absolutely no nonvolatile salt and limited concentration of volatile salts), so it is difficult to apply infusion in cases of limited sample.  When a nano ion source is used for infusion analysis, the sample consumption volume may be reduced to as low as a couple of microliters.

(16) What is difference between microLC/MS/MS and nanoLC/MS/MS?

The main difference is the flow rate used for LC/MS/MS analysis.  Usually microLC means the flow rate is run at 3 to 10 μL/min, while nanoLC indicates that the flow rate is run at 100 to 500nL/min.  As a result, microLC/MS/MS usually requires at least a 300 μm id C18 column which can be directly coupled with a standard ESI ion source for ESI/MS or ESI/MS/MS analysis.  In contrast, nanoLC/MS/MS requires 75-100 μm id C18 column coupled with a nano ion source for improving ionization under low flow rate.  Thus, microLC/MS/MS can provide relatively high throughput analysis (which costs less for each sample) compared to nanoLC/MS/MS analysis (which costs more per sample), while nanoLC/MS/MS analysis can provide considerably better sensitivity than microLC/MS/MS for protein identification, PTM characterization, etc.

(17) Can I run infusion ESI/MS analysis if I have very limited sample available?

The answer is yes. But you have to use nano ion source with extremely clean samples without any salts present which could suppress ionization of the analyte ions.

(18) Can LC/MS/MS be used for both protein identification and quantitation of my complex sample?

Yes, currently the most common techniques used for protein identification and quantitation by LC/MS/MS for complex sample are iTRAQ or TMT chemical labeling at the peptide level (Mol Cell Proteomics, 3(12): 1154-1169, 2004).  Other labeling technologies include enzymatic labeling with O18 (Analytical Chemistry, 73: 2836-2842, 2001) and SILAC (in vivo labeling with N15 and C13 (Mol Cell Proteomics, 1(5):376-86, 2002), and dimtheyl labeling (Nature Protocols, 4:484-494, 2009), as well as label-free approaches.  All can be used for both quantitation and identification when combined with LC/MS/MS analysis.  Currently, the facility offers the iTRAQ/TMT approach, dimethyl labeling, SILAC labeled sample analysis and label-free approaches (including spectral counting/emPAI, XIC and MRM).

(19) How can I interpret my Mascot results or Proteome Discoverer results?

Two database search tools have been heavily used by Cornell Proteomics Facility including Mascot and Proteome Discoverer. Mascot is a powerful search engine which uses mass spectrometry data to identify proteins from primary sequence databases.  Mascot integrates various proven methods of searching.  These different search methods can be categorized as follows:

  • Peptide Mass Fingerprint in which the only experimental data are peptide mass values
  • Sequence Query in which peptide mass data are combined with amino acid sequence and composition information.
  • MS/MS Ion Search using uninterpreted MS/MS data from one or more peptides.

Protein identification reports from our ESI/MS/MS or LC/MS/MS analyses fall into the last two categories.

For more information on how to interpret the search results, click here
For more information on scoring schemes, click here.
For more information on the result format and report, click here.

The Proteome Discoverer (PD) software from Thermo is another commonly used searching engine in Cornell MS facility for identifying proteins from the mass spectra of digested fragmented peptides. It compares the raw data from mass spectrometry to the information from the selected FASTA database. Specifically, the Proteome Discoverer application does the following:

1. Works with peak-finding search engines such as Sequest™ and Mascot to process all data types collected from low- and high-mass-accuracy mass spectrometry (MS) instruments. The peak-finding algorithm searches the raw mass spectrometry data and generates a peak list and relative abundances. The peaks represent the fragments of peptides for a given mass and charge.
2. Produces complementary data from a variety of dissociation methods and data-dependent stages of tandem mass spectrometry.
3. Combines, filters, and annotates results from several database search engines and from multiple analysis iterations.

If you are interested, you can learn more about the PD software through the following URLs. Tutorial videos for PD 1.4 are available online, click here.

The user manual of current PD 2.1 version can be downloaded in the following URL: click here

(20) Can I get the raw MS data files and do database searches by myself?

If you need MS raw data files for database search or for required data deposit for our publications, please contact facility staff and we will send you the raw data files through “Cornell.box.com” for you to download in a week after we receive your request message. Generally, the facility will provide the database search and report to you the final results. If you prefer to do search by yourself, the short answer is No. Unless you have owned the specific MS data analysis software and some specific search engine software in order to process the database query. That might not be practical for most users. There are two options to partially overcome that. One is that you can physically come to the facility for performing the database searching using the facility dedicated database searching computer. But this option is limited to the only raw data files generated from the facility. Also for this option, you have to attend a database training class and make sure the computer is available before you come to use it. There will be a use charge for using the facility computer and Mascot server. For details, please contact facility director. Alternatively, you can remotely access the facility designated computer for database searching. Again to use this option, you need to have database search training, have the remote desktop connection set up by our IT people and it will be limited to the raw data files generated by 4000 Q Trap.

(21) How can I get the MS or MS/MS spectra for making my PowerPoint presentation or for publication figures?

There are two ways to do this.  One is to ask the facility personnel to retrieve and send the spectra to you.  The facility will provide 1 to 2 spectra (as a screen shot) for each submitted project at no charge.  If you request more than that, we may add a time-based service charge for the additional work, such as polishing figures for presentations and publications.  The second option is that you can do it yourself.  You can either come to the facility or remotely access one of the facility's computers.  You can process the MS spectra generated from all three mass spectrometers in the facility.  Training is available for MS data processing.

(22) Are there training classes on the use of software for data interpretation and data processing?

We periodically hold training classes on the use of MS analysis software and database searching as well as MS data processing.  We also offer an annual 2-day MS technology workshop, which includes a full day focus on database searches and data interpretation.   For more information, please go to our Workshops and Training page.