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Small Molecule

FAQ - Proteomics

Frequently Asked Questions

How do I best identify my protein?
How much protein do I need for a successful analysis?

How do I best identify my protein?

Protein identification can be done either by enzymatic digestion of an in-gel sample followed by LC/MSMS analysis or direct n-terminal protein sequencing (Edman chemical sequencing). If the protein is n-terminally blocked (80% of eukaryotic proteins are naturally blocked) then digestion is the next logical step. Once digested the peptides can be separated and collected by off-line HPLC with UV detection (leading to Edman chemical sequencing of individual peptides) or the mixture analyzed on the mass spectrometers by LC/MSMS.

How much protein do I need for a successful analysis?

See the Protein Quantity Estimation section to start. Edman chemical sequencing needs 10pmol for high quality data, more for extended sequence runs. Keep in mind the initial yields for this technique is ~50%, with a repetitive yield around 92%. Typically samples can run 30 residues with ~100pmol sample loaded, depending on the protein and sample preparation. Mass spectrometry is several orders of magnitude more sensitive that Edman sequencing, thus it is possible to say we can get high quality data from as little as 10fmol protein. This amount, however, is not what was loaded on the gel, as each step in the sample handling process (reduction, alkylation, digestion, peptide extraction and concentration) incurs protein and peptide loss to surfaces. Maximizing quantity is always the first thing to do for any sample, and in some projects it is simply not possible to get more sample, so trace level analysis must be done with a subsequent loss of protein coverage and spectra quality to some degree. The degree of this impact is difficult to quantify, as each protein will behave differently and thus unpredictably at very low quantities. Also see the FAQ regarding silver stained gel spot or bands.

FAQ - Small Molecule

Unsure what ionization technique to choose? All samples will be analyzed by Electrospray (ESI), if ESI was unable to generate satisfactory results, direct EI/CI/FAB techniques can be carried out (please check appropriate boxes on the submission sheet if you would like EI/CI/FAB analysis after ESI failed. When in doubt, please ask your friendly staff before submission. Please visit JEOL Analytical Instruments website if you would like additional information on different types of ionization methods.
Do I need high resolution? High resolution is generally required for journal publications (≤ 1000 m/z). If you would like to have high resolution performed, on the submission sheets, please check the appropriate box for “High Res if Low Res Pass”. Please note that high resolution analysis will require a longer turnaround time and cost more than low resolution analysis.

What sort of turn around time can I expect? If you have urgent needs, please contact us for our rush service rates. Electrospray (ESI) low resolution data usually can be obtained within 2 business days while high resolution data usually can be obtained in less than 1.5 weeks. EI/CI/FAB low resolution data usually can be obtained within 5 business days while high resolution data usually can be obtained in less than 2 weeks. Please be patient, especially when the instruments are down for maintenance or the mass spec facility is experiencing unexpected high volume submissions.

What about MALDI analysis? Please contact us for current information.

How do I get to use the Open Access LC/MS and GC/MS systems? Contact us for a training session. It takes about 30 minutes. No one is allowed to use the system until they have taken this course. We can also arrange for you to gain access to the lab after hours once you are trained on the Open Access instruments.

Can I run all my samples by Open Access? The OA LC/MS instrument is equipped with an ESI ionization source. Your sample needs to be amenable to the method. While OA will provide satisfactory data in many cases, the diversity of chemistry undertaken in our department makes it impossible to give a detailed list of what will and will not work. However, the following types of materials are definitely NOT amenable: a) very volatile materials (ones you would normally submit for EI and CI). b) materials that will precipitate or decompose in polar solvents (many organometallics fit this category). c) OA GC/MS instrument is suited for semi-volatile and volatile samples with low molecular weight. Please consult with our staff for the appropriate instrument for your analysis.
What about different solvent systems? Our solvents are Water with 0.1 % of formic acid and Acetonitrile with 0.1 % of formic acid. Samples may be dissolved in most common VOLATILE organic solvents or water. Do NOT run samples dissolved in 100% DMSO and DMF, which will damage the instrument. Please consult our staff if you have any question regarding solvent use.
What about high resolution analysis? Our Open Access Agilent 6210 TOF LC/MS is capable of performing high resolution analysis (to 4 decimal places). Please note that for high resolution analysis, the sample should be pure.