LCMS Qtof analyses requirements
The primary purpose of LC-MS Sample Preparation is to remove any potential interferences, concentrate the analytes of interest, and make the sample compatible with the LC-MS system to ensure accurate and reliable data and not to contaminate the source of the mass spectrometer.
- Matrix Effects: Many samples, especially biological ones, have complex matrices that can interfere with the LC-MS analysis.
- Analyte Diversity: Different analytes might require different preparation techniques.
- Sensitivity and Specificity: Achieving the necessary sensitivity and specificity requires careful optimization.
Good sample preparation is fundamental to successful mass spectrometry and there is no one-size-fits-all method. Proteins must first be extracted from the sample in question and solubilized, before they can subsequently be digested to peptides for mass spectrometric analysis. However, some buffer components (in extraction and lysis buffers) can cause problems for down-stream mass spectrometry. Where contaminants such as inorganic salts and detergents are present in a sample they must be removed before submitting analyses.
The following requirements is necessary before samples will be analysed. Please aim the concentration of analyte in the range 10 micrograms per ml. When analyte concentration cannot be estimated, we may not be able to run your samples.
Contaminants Incompatible with Mass Spectrometry
Detergents, Polyethyleneglycol (PEG) and other Polymers
Detergents are frequently used during sample preparation in extraction and lysis buffers. The presence of PEG in a sample causes ion signal suppression on the mass spectrometer. Additionally, detergents
can reduce the efficiency of trypsin in the subsequent peptide digestion step, and they can be extremely difficult to remove from both the HPLC column and mass spectrometer.
• Reduce the risk of contamination by not using detergent to wash glassware
• Use mass spec grade solvents.
• Use good quality branded plastic consumables (eg. Eppendorf tubes)
• If possible, design your sample preparation to avoid using detergents.
Cell debris, insoluble material or particulates
These can become trapped in the chromatography system, causing
pressure irregularities and distortions in the chromatography. Filter your samples into the mass spec sample vial.
Cryoprotectants: DMSO, Glycerol, DMF (Not allowed)
These are viscous and can be detrimental to the chromatography system, and they can interact with tubing and other machine parts
Volatile buffers
Where possible you should avoid inorganic salts and non-volatile buffers in the final sample preparation steps e.g. Tris, phosphate. Non-volatile salts and buffers can form adducts, suppress ionization of your peptides, and affect the chromatography. Volatile buffers such as ammonium bicarbonate or TEAB will leave no residue when the sample is dried down prior to final re-suspension for loading onto the mass spectrometer and should be used in preference