It is currently possible to analyze thousands of proteins in a single sample using mass spectrometry (MS) and a database of predicted protein sequences, referred to as ‘bottom-up’ proteomics. With this technology, you can measure protein levels and interactions. Also, you can examine changes in post-translational modifications (PTMs) and isoforms (in an unbiased manner). Working with an obscure organism where antibodies aren’t available? No worries. You can perform more sensitive and unbiased experiments without ever buying or developing one. Looking at a protein phosphorylation site that isn’t reported in the literature? You can do that too.
Basically, anyone can visit his or her local proteomics facility, give them a sample, and (hopefully) get back a list of proteins to try and interpret. But if you listen to people who run mass-spec facilities, you will quickly learn that the majority of molecular biologists do not understand the requirements for good sample preparation. Many treat the mass spec as a black box capable of generating publishable data from sub-par inputs. Nothing could be further from the truth.
What Constitutes a “Bad” Proteomics Sample, you Ask?
Generally, it’s one of three things:
- the presence of biological or chemical contaminants that make LC-MS data acquisition and analysis impossible or very difficult
- a group of samples (e.g., biological replicates) that have not been prepared in a reproducible way, or
- a nonexistent “sample” that runs like a blank.
How can you Avoid these Pitfalls?
Usually, the first step of a proteomics experiment is to digest your protein sample (a purified protein, an immunoprecipitate (IP), a protein extract or an enriched fraction) into peptides with a site-specific protease, like trypsin. You then clean them up (desalt) and load the peptides onto a high-performance LC setup, where they are fractionated on a column and then detected by electrospray ionization in the mass spectrometer1. From there, the mass to charge (m/z) ratio and abundance of the peptides are measured. Since the human genome has been deciphered, you can computationally map peptides back to their parent proteins and infer differences across samples and conditions.
In general, you will use one of three sample prep methodologies. These are in-gel digestion2, in-solution digestion, or filter-aided sample preparation (FASP)3. I’ll give the pros and cons of each, but my not-so-humble opinion is that you should use FASP for basically all sample types as it is universal, fast and easy for newcomers, fairly high-throughput, and generates high quality proteomics samples provided that you do your part.
In-gel Proteomic Digests
- Uses SDS-PAGE, which provides good resolution and is familiar to most molecular biologists
- Solubilizes most proteins in the proteome by using SDS
- Allows for limited analysis or identification of unknown proteins by cutting specific bands out
- Resolves contaminating proteins and molecules through electrophoresis
- Low throughput and is laborious
- Digesting gel bands is sometimes inefficient
- Prone to biological contamination
In-solution Proteomic Digests
- In-solution digests have higher throughput than in-gel digests
- Can identify all peptides in solution, depending on complexity
- Reduces risk for contamination (no handling of acrylamide pieces)
- Utilizes chaotropic agents in the protein prep, which may not solubilize proteins as well as SDS does
- Requires protein precipitation to remove substances that interfere with trypsin digestion
- Nucleic acids, phospholipids, and other non-peptide biological molecules are not removed during the tryptic digest, leading to downstream problems in LC-MS setups
- Solubilizes most proteins in the proteome by using SDS
- Identifies all peptides in solution, depending on complexity
- Compatible with many sample types
- Easy to learn, even for beginners
- Same throughput as in-solution digestion
- Resolves contaminants by using buffer exchange in a molecular weight cutoff filter. All steps in the same tube.
- Trypsin and other proteases stay behind in the filter; while your peptides spin out when digested
- DNA, RNA, lipids, etc. are largely removed, making the sample cleaner for downstream MS analysis
- Exhibits some sample loss through filter binding
- Filters are more expensive than gels or microcentrifuge tubes
The Importance of Reagents in Proteomics
Coupling FASP with good technique will take you a long way. To start, always use HPLC or LC-MS grade reagents, and don’t stick your dirty pipettes into them. Ever. Pour stock solutions out. This includes water, methanol, acetonitrile, acids and anything else you can think of. Make all of your solutions in LC-MS grade water. You will be using 4% SDS to solubilize your sample, so avoid nonionic detergents like NP-40, Tween, and Triton X-100. Detergents like these ionize in the mass spec like crazy and can trash an LC column. This is guaranteed to “amuse” your facility manager and cost you a cool $500.00 for column replacement.
When using organics such as acetonitrile, pour them out into really clean glass vials. If you use plastic tubes with concentrated organics, you will probably strip polymer into your sample. If you’re using spatulas, pipettes, and other standard equipment to make solutions, be sure it is all ridiculously clean by using 70% ethanol and KimWipes. It is also best to have dedicated reagents, glassware and plastics only for mass spec sample prep.
Avoiding Biological Contamination and Sample Loss
It’s best to track the concentration at every step and do your best to normalize your samples before they go into sample vials. This means that the same amount of total protein should be used for trypsin digestion (using a protein assay like BCA or Lowry). And also that the same amount of digested peptide should be put on the desalting column. To determine peptide concentrations, use A280 (like a nanodrop) prior to performing the desalting step. You should also avoid excessive sample handling (pipetting, opening tubes, etc) to reduce loss and the chance for contamination.
It is almost always a good idea to use low retention microcentrifuge tubes. They bind less protein than the normal variety. Clean your bench well with ethanol and change your gloves often. Keratins are all over your skin and in dust and are one of the most common biological contaminants in MS experiments. Pay careful attention to the way you handle tubes; avoid working over top of them or leaving them open. Lastly, if you’re working with human cell lines, wash them well with PBS prior to harvesting. BSA is an extremely prevalent protein in FBS and can dwarf signals from your proteins of interest if not removed prior to sample prep.
Whew! That was a lot. Maybe most importantly, you should have everything set up for sample prep day. And plan your experiments carefully with your facility manager before going to the bench. It will ultimately save you time and money. Keep calm and mass spec on for great proteomic results!
1. Steen, Hanno, and Matthias Mann. The ABC’s (and XYZ’s) of peptide sequencing. Nature reviews Molecular cell biology (2004) 5.9: 699-711.
2. Shevchenko, Andrej, et al. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nature protocols (2006) 1.6: 2856-2860.
3. Wisniewski, Jacek R., et al. Universal sample preparation method for proteome analysis. Nature methods (2009) 6.5 : 359.