Protocols
Sample Preparation
Sample Concentration
Use the following guidelines for minimum sample concentration. A sample volume of at least 3 – 5 µl is desirable. Larger volumes are necessary for more dilute solutions. Solvents must be clean (salt and additive free) to obtain optimum signal (see Solvents and Buffers Suitable for Electrospray).
- 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).
Solvents and Buffers Suitable for Electrospray
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 (HPLC grade or better), methanol, acetonitrile, formic acid, acetic acid, trifluoroacetic acid (lowest concentration possible), <=10 mM ammonium acetate, <=10 mM ammonium bicarbonate,<=10 mM ammonium formate, and <=2% triethylamine (good for oligonucleotides).
Gel Plugs for Protein ID: Proteins and peptides separated on one or 2-D gels for subsequent identification analysis by LC/MS and MS/MS (Q-Tof2 and QSTAR XL) 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 (or arm covers), 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 (available 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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