Protein sample preparation for Mass Spectrometry

Protein sample preparation for mass spectrometry

There are a number of factors to consider when preparing a protein sample for mass spectrometry. Designing a sample preparation strategy should account for the following: source and physical properties of the sample, abundance of the target proteins, as well as complexity and cellular location of the proteins. Understanding these elements of your sample will narrow the options for mass spectrometry procedures. When working with complex samples, it may be necessary to use specialized techniques for selecting target proteins. This may require workflows that incorporate:

• Optimized cellular lysis
• Subcellular fractionation
• Depletion of abundant proteins
• Enrichment of select proteins
• Mass tagging tools

Each of these steps can play a key role in producing accurate results in protein detection or isolation.

Lysate preparation

Physical lysis is a technique used to disrupt cells and extract cellular contents. It requires specialized equipment and protocols, as there is often significant variability in the apparatus. This can look like a difference in sonication settings, or even different pestles.

Cell lysis disrupts cellular compartments, activating endogenous proteases and phosphatases. This makes it important to protect extracted proteins from degradation by the activities of these enzymes. This can be done by adding a protease and/or phosphatase inhibitor to the lysis reagents.

There are some limitations when performing cell lysis that may require alternatives or further processing techniques. Lysis is typically not conducive to small sample volumes or high-throughput sample handling. Physical lysis methods on their own will not solubilize membrane-associated proteins.

However, reagent-based lysis methods can lyse cells, while also solubilizing proteins. By utilizing different buffers, detergents, salts, and reducing agents, cell lysis can be optimized based on the respective cell type or protein fraction required.

To maintain integrity of the target protein, some detergents may need to be added and later removed to properly study the proteins. Doing so will maintain the long-term stability of the extracted protein.

Depletion and enrichment

The objective of depletion and enrichment strategies are to remove high abundant proteins or isolate target proteins in the sample.

Depletion is often used to reduce the complexity of a biological sample (such as blood or serum, which contain high concentrations of albumin and immunoglobulins). Immunoaffinity techniques such as immunoprecipitation, co-immunoprecipitation, and commercial kits are all available to perform depletion on a sample. The only significant drawback to this technique is that abundant proteins often bind to other proteins, resulting in the depletion of complexes with low-abundance proteins.

Protein enrichment isolates subclasses of cellular proteins. This can be achieved using a number of techniques that identify; unique biochemical activity, post-translational modifications (PTMs), or spatial localization within a cell. Proteins can also be enriched using enzyme class-specific compounds or cell-impermeable labeling reagents that selectively label cell surface proteins.

Separation of distinct subcellular fractions can be utilized as a method of enrichment. This can be achieved via physical disruption techniques, detergent-buffer solutions, and density gradient methods. This type of separation is ideal in circumstances when attempting to pull a hydrophobic membrane protein from a hydrophilic protein.

Lastly, density gradient centrifugation is another technique that is ideal for isolating intact nuclei, mitochondria, and other organelles prior to protein solubilization.

Dialysis and desalting

In order to ensure a sample is compatible and optimized for digestion and analysis by mass spectrometry, further steps like dialysis and desalting may be required.

Mass spectrometry measures charged ions, meaning salts should be removed prior to MS to minimize their detection (especially sodium and phosphate salts). Dialysis and desalting allow buffer exchange, as well as the removal of small molecules to prevent interference with downstream processes.

Protein denaturation and digestion

Digestion can be performed both in-solution or by 1-or-2-dimensional gel electrophoresis. Using one technique over the other comes down to two primary factors - sample amount and complexity.

When performing in-solution digestion, proteins are denatured with strong chaotropic agents, followed by a reducing agent. These denatured, reduced proteins are then alkylated to irreversibly prevent reforming of disulfide bonds. The alkylated proteins are then digested by endoproteinases, which hydrolytically break peptide bonds to fragment proteins into peptides.

In-solution digestion is ideal when working with a small sample, as the peptide extraction from in-gel digestion can result in significant peptide loss. In-solution digestion is also best for samples with low-to-moderate complexity, where detergents would negatively impact the sample. When it comes to duration and automation, in-solution digestion is preferred. Not only is it performed more rapidly, but it is also commonly automated.

Protein separation by gel electrophoresis employs SDS polyacrylamide gel electrophoresis to denature and separate proteins in a sample. After electrophoresis, the protein bands are visualized and then excised from the gel. The gel plugs can then be reduced, alkylated and digested. Peptides are then extracted from the gel matrix, which can then be prepared for MS analysis.

One benefit of in-gel digestion is that it combines protein denaturation with separation, visually indicating the relative abundance of proteins in the sample. Peptide extraction also removes a great deal of the detergents and salts, though this can affect peptide recovery.

Peptide enrichment

In the final phase of sample preparation for mass spectrometry, enrichment of target peptides is performed. This phase, along with sample clean-up, is required for successful analysis of low-abundance proteins or identifying post-translationally modified peptides.

Enrichment is performed by affinity purification using specific antibodies or ligands, which will selectively bind to the target compounds.

After peptide enrichment, salts and buffers can be removed using affinity columns or detergent-precipitating reagents. Concentrators can also be used in dilute samples, which come in a variety of molecular weight cutoff ranges (MWCO).

At this time, the purified peptide sample is ready for the final preparation for MS analysis. If operating LC-MS or LC-MS/MS analysis, the correct choice of mobile phases and ion-pairing reagents is crucial to achieve good LC resolution for quality analytical results.

Reasons that protein analysis goes wrong

There are a number of factors that can lead your sample preparation astray, leading to improper mass spectrometer analysis. One of the more common roadblocks to run into with mass spectrometry are complications with the analysis being unable to identify the amino acids. Here are the most common errors behind this mistake:

• Improper digestion of protein
• The hydrophilic/small nature peptide passes through the back stage column with the salt and cannot be analyzed
• Poor fragmentation - peptides are hydrophobic or too large, and cannot be removed from the column, or are too big for analyzing via MS
• Ways of peptide fragmentation cannot be analyzed. Several spectra are unexplained in the investigation

Things to avoid when preparing a protein sample

Ionization and Sample Concentration

Ionization techniques used for large molecules often work well when the sample mixture contains equal amounts of constituents. When this is imbalanced you may run into problems. For samples with a high range of protein abundance, the more abundant proteins tend to suppress signals from less abundant ones.

Simplify Your Sample

Analyzing the mass spectrum of a complex mixture is already a challenge due to the large number of components. This can be further exacerbated by enzymatic digestion of a protein sample into a large number of peptide products. A successful analysis depends on a clean sample with limited complexity. It’s important to minimize any complexities that would cause suppression of ionization or under-sampling of eluting peptides

Complex Samples and Enrichment

One challenge of mass spectrometry is that it can be difficult to detect trace-level proteins when they are present at much lower concentrations than other biological products. This can be combated by overloading the column, however this is at the expense of ionization suppression and MS detector saturation. Enriching samples by depleting product protein is a long-term goal in the field, which has shown some success.

Pros and cons of mass spectrometry

Mass spectrometry presents distinct advantages as an analytical technique:

• Increased sensitivity over most other analytical techniques
• A mass-charge filter reduces background interference
• Excellent specificity utilizing fragmentation patterns to identify unknowns or confirming the presence of compounds
• Information on molecular weight. Identify unknown compounds
• Data about the isotopic abundance of elements

However, there are a number of disadvantages:

• Fails to distinguish between optical and geometrical isomers 
• Fails to differentiate between positions of substituents (in o-, m-, and p- positions)
• Limitations on identifying hydrocarbons that produce similar fragmented ions

Even with these risks and limitations detailed above, the greatest challenge in mass spectrometry will be the variations in sample type that you will come across. Each sample will have a unique makeup, and may even be received at varying levels of sample preparation process.

Understanding how to work through these parameters to produce samples that are compatible with the mass spectrometer is key to your success. This will require flexibility, creativity, and an understanding of the many options we covered on sample preparation. For more information on analytical testing, visit Avantor for all of the latest developments in chromatography and mass spectrometry.