Frequently Asked Questions
What are the optimal conditions for sample concentration and volume when preparing for electrospray analysis?
Use the following guidelines for minimum sample concentration. A sample volume of at least 3 – 5 µl is desirable, more for dilute solutions. Depending on the sample, a concentration below that indicated may be sufficient. Clean, salt- and additive-free solvents are critical to obtain optimum signal.
- Peptides (<5 kDa): 1 – 50 pmol/µl
- Proteins (=5 kDa): 5 – 50 pmol/µl
- Oligonucleotides: 10 – 100 pmol/µl
- Enzyme digests: 1 – 20 pmoles—injected onto LC/MS columns
If necessary, samples containing high salt may be submitted for analysis by C18 RP LC/MS trap cartridge clean up and online infusion. In this instance it would be more convenient and assay efficient if additional sample was available (10 – 20 µg) but this is not requisite.
What solvents and buffers are suitable for performing electrospray analysis?
The mass spectrometer detector is very sensitive and will "see" all ionizable substances in buffers and mobile phases. One must avoid non-volatile salts, in particular: phosphate, sodium (keep to less than 1 mM) and detergents (keep to less than 0.01%, avoid or remove if possible). A detergent concentration of 0.1% variably affect signal response = 60% dependent upon the detergent used. At 1% most detergents eliminated the protein signal.
Suitable mobile phases, buffers and solvents should be formulated from the following: water, methanol, acetonitrile, formic acid, acetic acid, trifluoroacetic acid (lowest conc. possible), = 10 mM ammonium acetate, = 10 mM ammonium bicarbonate, = 10 mM ammonium formate, and = 2% triethylamine (good for oligonucleotides).
How do I handle and stain one and 2-D gels and excise protein bands/spots for analysis on the micromass q-tof or applied biosystems QStarXL mass spectrometers for protein identification and/or map coverage?
Proteins and peptides separated on one or 2-D gels for subsequent identification analysis by LC/MS and MS/MS (QTof2 and QStarXL) need very careful handling. All labware, solutions and reagents used for preparation of gels for electrophoresis and storage of protein/peptide solutions need to be free of any contaminants in particular, keratins. At a minimum, researchers should wear gloves, long sleeve lab coat and if possible use a laminar flow workstation with HEPA filters to utilize and keep a clean work area. For MS analysis the gel resolved proteins should be stained with a sensitive colloidal Coomassie Blue stain for proteomics applications. Intensely stained bands or spots of interest should be excised in as small a band or plug, respectively, as possible and transferred to rinsed microfuge tubes for storage in 10% HAc, 50% MeOH solution prior to submission for analysis.
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