Mass Spectrometry & Proteomics

Before sample submission

Contact the MS team. We advise you on the best analytical methodology for your specific application.

Instructions for sample delivery

One of the most important steps to achieve a successful result in mass spectrometry analysis is a well-planned experiment involving an adequate sample preparation for each MS analysis. Specially, for proteomic experiments is strongly recommended to contact with MS team before starting it. Here you have some general instructions, but do not hesitate to contact us for any doubt.

General features

1. Work in a clean environment.
2. Avoid keratin contamination by using gloves and lab coat.
3. Do not use autoclaved material.
4. Wash all the material with milliQ water and MeOH (high quality) and let them dry before use.
5. Use LC-MS grade reagents.
6. If possible use low-binding eppendorf.
7. Use simple and well-recognized tube labeling. We recommend using consecutive numbers.

Samples for in-gel digestion

1. When possible run a 0.75 mm thickness gel.
2. Use clean recipients for staining. Close them during staining period to avoid contamination.
3. NEVER use recipients that were previously used for Western blot.
4. Use a mass spectrometry compatible protocol for gel staining.
5. If you use fluorescent stain do not cut the bands manually by using a transilluminator.
6. Cut only the stained region. The higher protein concentration and the lower acrylamide presence, the better protein identification.
7. When possible cut the gel spots or bands in a laminar flow hood to avoid keratin contamination.
8. Store bands at 4ºC without water or other solvents.

Samples for in-liquid digestion

1. There are some reagents not compatible with mass spectrometry. Below are listed the most commonly used and not compatible with LC-MS. Please tell us the reagents you use for sample preparation.
   • Igpal/NP40 is almost impossible to remove and shows large polymers along all the chromatogram, so we will suggest changing it for N-octyl-β-glucopyranoside or avoiding any detergent if possible.
   • The RNAse is a protein that could mask the proteins of interest.
   • When possible avoid glycerol.
2. For IP experiments a negative and a positive control are needed to find out nonspecific interactions. We also recommend running a SDS-PAGE gel with 10% of the sample to have an idea how the IP works.
3. The amount of sample required for protein identification depends on sample complexity and purity. For complex samples at least 1nM is required.

Intact proteins

1. An efficient ionization (ESI or nanoESI) of proteins depends on their length, and physicochemical properties (pKa, solubility, structure and hydrophobicity). It is possible to work under denaturing or under non-denaturing conditions
   • Denaturing conditions: proteins are solubilized generally in a 1:1 mixture of acetonitrile/water or methanol/water and 1% formic acid is added to enhance ionization (all solvents must be MS quality). Depending on their purity, proteins are analyzed by infusion or by LC-MS. 1μM to 10 μM protein concentration is required (volume: 10-100 μl). Low dynamic ranges are normally achieved and pre-purification is therefore required. Non-volatile salt removal is necessary (Zip-tips can be used). Maximum non-volatile salt accepted in the case of LC-MS experiments is 150 mM. Infusion experiments require complete salt removal.
   • Non-denaturing conditions: proteins or non-covalent protein complexes are solubilized in NH4OAC (10mM-1M). Salt removal is imperative and is done normally by dialysis or with Biospin columns. 5μM to 50 μM protein concentration is required.
2. Top-down (MS/MS experiments) can be performed on intact proteins to localize modifications or confirm amino acidic sequence. Protein length is limited to 30 kDa to perform MS/MS in the gas phase with the aim of entirely sequencing the protein. Middle-down strategies are otherwise used. Denaturing conditions are used in this case. 5μM to 10 μM protein concentration is required (100 μl).
3. Top-down can also be used for protein identification (up to 100-150 kDa). In this case protein coverage is low (this strategy is very much in underdevelopment with respect to the classical bottom-up approach (proteomics) although useful for protein isoform identification )

Sample submission/reception

There are two ways to submit samples for MS analysis:

1. Submission to the technician-run service by filling in the sample/s request form (available here or in the MSC lab).
2. Self-service (open-access) systems. Only for researchers qualified and trained to operate the mass spectrometers.
   If you plan to use the MS service often and would like to be considered for training, please contact Dr. Marta Vilaseca.

Custody and maintenance of samples

When filling the request form, your sample or set of samples will be identified by a code number (apart from the users label). Depending on the analysis requested it will be classified in the corresponding "to-run" folder for each instrument.

The sample will be also classified and kept in the conditions specified in the request form.

If preferred you can keep your sample and bring it a day before the beginning of analysis (following scheduled dates).

Time of response for an analysis

There is a calendar (contact the facility manager) for each instrument and submissions will be ordered by time of entry (IRB Barcelona groups are given preferential treatment).

Given the diversity of applications, some of which require only fast analysis while others require longer analysis time, the calendar will be organised as follows:

• Users can reserve a week in a specific instrument for long application (maximum 2 full weeks per user, depending on demand).
• Between long time applications we always reserve a week to perform shorter analysis in order to avoid delays.
• Mondays (or a pre-specified day) are reserved for the maintenance of the equipment and set up of the required technique pretended to use that week in each instrument.

Results delivery

Results performed by MS staff will be given in printed format (spectra and report specifying analytical conditions). Digital format data can also be kept if requested.

If further processing of your results is required a computer will be reserved in the facility for this purpose.

Our future goal is that users can also access data via a secured network. Data will be transferred to a common-server and back-ups will be performed automatically by the ITS Department. Data will be kept for a maximum of two years.

Contact

Mass Spectrometry Request Form

e-mail: marta.vilasecairbbarcelona.org, marina.gayirbbarcelona.org

Telephone: +34 934039815