Once you submit the form, you will receive an email acknowledging the receipt of your application.
After that, we will generate an invoice and will send it back to you for the costs of the mass spectrometry services. It usually takes 3-5 working days to generate the invoice.
After receiving the invoice, you can make the payment according to the payment options (online, DD or direct cash) provided in the invoice.
Once the payment is made, share the transaction details (reference number, date of payment) with us so that we will generate a payment receipt and will send it back to you.
After making the payment, you can submit the samples at a convenient time after consulting with our facility (by email or telephone).
In-solution protein samples: We prefer to
have a concentration of 1 mg/mL and 100 µl is
required (i.e., 100 µg in 100 µl). If the
concentration of your sample is less than 1 mg/mL,
let us know in advance. If you have
samples to be compared (for relative protein
quantification ), all samples should be in
uniform concentration and volume. In this case, we
prefer the samples to be in aqueous solution of
ammonium bicarbonate buffer (50 mM) having pH around
8, the optimal pH for trypsin activity.
Gel bands: A visible band with Coomassie
stain is sufficient. However, if the band is
extremely faint, there is a likely chance that we
may not get any identification. So, we suggest you
load the maximum possible amount of protein solution
into the polyacrylamide gel matrix so that we get a
fairly stained gel band.
Molecular weight determination: We prefer to
have a concentration not lower than 1mg/mL and 20-50
µl is required. In this case, the buffer composition
is not highly critical, as long as it does not
contain detergents or more than 5% glycerol.
The common contaminants, even in small quantities,
can mask important peaks in your MS data and have a
huge impact on the final results. The usual
contaminants in MS include keratin, polyethylene
glycol, polypropylene glycol, phthalates, ions,
polymeric detergents (tritons, tweens etc.) and
siloxanes.
Controlling LC/MS contaminants
Always use filtered de-ionized water.
Perform sample processing in a laminar flow
hood.
Any glass container, flasks or tubes used to
prepare buffers or samples must be thoroughly
cleaned before use. Never use detergents or
dishwasher to clean the beakers and glass plates.
Always rinse glassware using organic solvents (70%
ethanol or 70% methanol) and then filtered deionized
water.
Always wear particulate-free, powder-free,
nonâ€latex gloves and rinse them occasionally as they
readily attract dust or hair particles. Avoid skin
contact with the gloves on.
Refrain from using deodorant and other cosmetic
products during sample preparation. Siloxanes,
present in these compounds can cause MS
contamination under certain conditions, such as nano
flow.
Do not store liquids in plastics (source of
phthalates). Also, never use parafilm or other
plastic films to cover solvent reservoirs.
Visually inspect the vials, beakers, flasks,
and tubes and make sure that they do not contain
contaminants.
Detergent removal spin columns are available, for
example, PierceTM Detergent Removal Spin
Column which can be used to remove the detergents
before sending in your samples to the facility.
Another way to remove detergents, salts, and other
LC/MS incompatible things from samples is to do
'gel-assisted' proteolysis (Here, we trap the
protein solution in a polyacrylamide gel matrix and
wash out detergents, salts, and chaotropic agents,
and perform in-gel digestion). In this case, run the
protein sample approx. 1-2 cm into the gel. DO NOT
allow the proteins to resolve in the gel (this is a
sample clean-up strategy only). Then, PRECISELY cut
out the entire sample up to the sample front (only
the stained portion of the gel) - any excess gel
will lead to background noise. Then, send us the
diced gel band. We will perform in-gel digestion and
subsequent LC/MS analysis.
Only volatile salts (e.g., ammonium
bicarbonate, ammonium formate, ammonium acetate,
triethylammonium bicarbonate (TEAB)) are compatible
with mass spectrometers. Non-volatile salts and
additives (e.g., sodium (Na+), potassium (K+), or
phosphate) can precipitate at the high-organic end
of the solvent gradient. This will block the columns
and MS source, resulting in painstaking cleaning
procedures. If your sample contain non-volatile
salts or additives, perform dialysis, or use salt
removal kits, for example c18 Zip Tip (Millipore,
#ZTC18M096, 96 Pk), or a centrifugal filter with an
appropriate molecular-weight-cut-off (MWCO)
membrane. We suggest performing buffer exchange
using ammonium bicarbonate buffer (50 mM), having pH
around 8, using Amicon Ultra 0.5 mL centrifugal
filters with 3,000 MWCO for this task (Millipore,
#UFC500324, 24 Pk).
Another way to remove detergents, salts, and
other LC/MS incompatible things from samples is to
do 'gel-assisted' proteolysis (Here, we trap the
protein solution in a polyacrylamide gel matrix and
wash out detergents, salts, and chaotropic agents,
and perform in-gel digestion). In this case, run the
protein sample approx. 1-2 cm into the gel. DO NOT
allow the proteins to resolve in the gel (this is a
sample clean-up strategy only). Then, PRECISELY cut
out the entire sample up to the sample front (only
the stained portion of the gel) - any excess gel
will lead to background noise. Then, send us the
diced gel band. We will perform in-gel digestion and
subsequent LC/MS analysis.
To,
Mass Spectrometry Core Facility, Second Floor (Extension: 536)
Rajiv Gandhi Centre for Biotechnology (RGCB),
Thycaud Post, Poojappura,
Thiruvananthapuram - 695 014, Kerala, India
The samples should be sent in such a way
that it reaches RGCB on a weekday (Monday - Friday).
The results of the analysis would be communicated in
the form of Excel sheets with data including protein
accession number, protein name, peptide count, unique
peptide count, molecular weight and abundance.
Where the position of a PTM is known/suspected,
these can be scanned for directly using the analysis
software. Enrichment strategies is needed for certain
modifications (e.g., Phosphorylation). Please discuss
PTM analysis with the facility staff.
Yes. The species details are always preferred as it
would lead to precise data comparison using
appropriate database. In cases where the species
(organism) of interest is not a model or widely
studied organism, the user should either provide a
protein database (in .fasta format) or indicate a
publicly available database for searching.
A clear stained single gel band (usually) contains
several proteins of (almost) the same molecular
weight. Many of these proteins might be below the
detection level of the staining method employed. The
sensitivity and resolution of LC/MS is far superior
compared to that of coomassie and silver staining.
There can be several reasons for this. The most
common ones are given below.
Your protein of interest might not be present in
sufficient amounts to be detected among other
proteins (of (almost) the same size) in the gel band.
Your protein of interest might not have sufficient
number of cleavage sites for trypsin, our protease.
This usually happens for low molecular weight
proteins.
Your protein of interest is not present in the
database. This usually happens when the species
(organism) of interest is not a model or widely
studied organism.
The biological variation of the samples is usually
large, compared to the technical variation. Therefore,
we suggest having multiple biological replicates for
each sample (a minimum of two). We always do two
injections per sample (two technical replicates).
Once we receive the samples, usually it takes 2-3
weeks to digest the samples, and perform LC/MS
analysis and data analysis. But, depending on the
number of samples and the availability of the LC-MS
instruments, this time can vary. Anyways, we will
notify you if there is any delay in processing the
samples.
Contact
Rajiv Gandhi Centre for Biotechnology (RGCB), Thycaud Post,
Poojappura, Thiruvananthapuram - 695 014, Kerala, India +91-471-2529400
| 2347975 | 2348753 +91-471-2348096
webmaster@rgcb.res.in