Sample preparation for mass spectrometry
What do you mean "I cannot just put the sample in the machine.."?
Mass spectrometry is a tricky science. It is incredibly powerful and can bring a lot of added value to most research projects. However, in order to benefit from the full potential of the analysis, one has to be very careful during the sample preparation stage of the experiment. Because no, you cannot just put the sample in the machine in any way! Such a sophisticated machine needs that your sample is prepared the right way, with the right tools and the right techniques. Only then will you get meaningful results. And because we are all about helping people, we will share our secrets to you. So, here are some tips for sample preparation for mass spectrometry!
1. Use the right solutions grade
You have probably noticed that some of the solutions that you buy come in different "quality", or grade. Since you are to analyze your sample by LC-MS, you are required to use LC-MS grade solvents in order to get the best possible conditions for the analysis. These solutions may be expensive, but they are a central part of the sample preparation and should be treated as such!
2. Pay attention to what you feed on the machine
Not every chemical is welcome when performing LC-MS analysis. For example, common detergents such as Triton X-100 and SDS can be very hard to deplete from your sample and can compromise sample analysis by creating ion suppression (click here for a good review on the subject). When the use such molecules is unavoidable, special preparation techniques, such as the Amicon adapted enhanced FASP (designed at PSB!) can help to accurately deplete the unwanted molecules from you sample. In all cases, one should always verify with experts in MS if their preparation protocol is suitable for MS analysis.
3. Use low binding tubes and pipette tips
Some analytes (such as proteins and peptides) have a tendency to bind to untreated tubes. To prevent this when preparing samples, we only use low-binding tubes (such as Protein Lobind tubes from Eppendorf). These tubes have been shown to reduce protein and peptide loss by a huge factor. In addition to low-binding tubes, we always use filter pipette tips to prevent contamination of the samples by molecules that could be stuck on the pipette end. And finally, we never use autoclaved plastic material, either tips, tubes, plates or pipettes. The heat and the pressure during the autoclaving process "fragilizes" the plastic, which leads to a contamination of the samples by polymers that hinders the MS analysis.
4. Test the pH of your solutions
The purpose of liquid chromatography is to separate the analytes of a sample to provide better sensitivity and quantification accuracy. And since it relies on the chemical properties of your analyte of interest for the separation, it is really important to make sure that the pH of your preparation solutions is the what it is supposed to be. Always double check your solutions!
5. Purify your analyte with the right tools
In mass spectrometry, it is really common to isolate the analyte of interest from the rest of the unwanted molecules. We do this to maximize the sensitivity of the machine for the molecule to quantify. To do so, one can use different affinity based methods such as solid phase extraction (SPE), or liquid-liquid extractions and simplified liquid extraction. Using the right method can make a huge difference in the sensitivity and the specificity of the analysis. There are several free online tools (such as the SPE method development tool from Phenomenex) to help you to design the best purification method for your molecule of interest; use them to save you time (and money on bad experiments!).
6. Use internal standards
This will make your standard curve's R2 go from 0.679 to 0.999! The use of a proper internal standard helps to correct the variations in the sample preparation method and leads to a more precise quantification of your molecule of interest. Be it a labeled version of your molecule or a different isotope, it can turn a non-significant change into a highly significant one. Never try to save money by omitting internal standards: it can make or break your experiments!
7. Use only high quality products
This one may seem pretty obvious, but we'll write it here anyways : you should only use good quality products when doing MS experiments (or any experiments!). Using a cheap product to conduct a highly sophisticated experiment makes no sense. This means that saving a 100$ on a cheaper version of a working product is just not worth it. For example, you should not try to get the absolute cheaper magnetic beads to do an IP-MS experiment in the hopes of identifying the interaction partners of your protein of interest. If you do, you may as well get poor enrichment of your bait and drown its interaction partners in high background protein levels.
As a final note, we are not against you saving money! We only state that if you choose a bad product, you may compromise your MS experiment, which will result in you losing a lot more than you saved with that cheap product.
Sample preparation is one of the most important part of a successful MS experiment, along with the instrument and the software. It is a central part of the method development of a MS project and should be treated as such. This article was meant to provide general guidelines on the sample preparation and to raise awareness on some key points when you choose to prepare your samples in your lab. If you have any questions on sample preparation or method development, contact our experts right away!