In a webinar from Thermo Scientific, Katie Southwick describes applications for analyzing intact proteins using the Q Exactive platform (Thermo Scientific). In contrast to top-down proteomics, liquid chromatography and mass spectrometry (LC-MS) in biopharma protein applications focuses on characterizing less complex samples. Often, these experiments target recombinant proteins, antibodies or a single protein of interest. These applications are also useful for quantifying known proteins or identifying impurities.
For intact protein analysis, the preanalysis steps are critical to success, since sample handling and storage affect the experimental outcome. Southwick recommends that peptides or protein samples stored as solids be resuspended in the buffer just prior to the analysis. For liquid samples, she recommends storing samples in a concentrated form in 20 mM ammonium acetate plus detergent. For both solid and liquid samples, she advises storing in LoBind protein tubes or glass vials at –80 ℃ while avoiding multiple freeze-thaw cycles.
Sample cleanup enhances sensitivity by removing detergents, salts and other adducts that suppress ionization by creating spectral interferences. Looking to specific cleanup protocols, Southwick makes some recommendations, while noting that the precise cleanup method is dependent on the particular sample of interest. She concludes that the BioRad Bio-Gel P-6 and P-30 and the Thermo Scientific Zeba Spin Desalting Columns work well for desalting and promoting buffer exchange. To remove detergents, the HiPPR Detergent Removal Spin Column for samples up to 100 µg/ml is a good option. The Thermo Scientific Slide-A-Lyzer G2 Dialysis Cassettes are optimal for samples with smaller volumes.
Next, Southwick points out that one way to troubleshoot the sensitivity of a particular analysis is to introduce a clean standard using the given reaction conditions. If the clean standard is detected at the correct spectra, but the target sample is not, there is likely a problem with the target sample. In this scenario, an ion trap should also detect a clean sample. Researchers should choose standards that have similar molecular weight as the intact protein. Recommended clean standards for the Q Exactive mass spectrometer include ubiquitin (8.5 kDa) apomyoglobin, (17 kDa, Sigma A8673), carbonic anhydrase II (30 kDa, Sigma C2522), enolase (46 kDa, Sigma E6126-500 UN) and mAb (150 kDa, Waters 186006552). Researchers should prepare standards as a 1–2 ml of a 10 pmol/µl solution in 50% ACN and 0.1%FA.
Now turning to the various optimization controls within the Q Exactive platform, Southwick begins with the ion source interface. She advises that running the capillary temperature at a high temperature of 275–325°C will improve desolvation. Another important optimization is to test the effects of an increasing S-lens RF. Typically, an RF of 60–70% is optimal, since the higher the S-lens RF is, the easier it is to transmit large ions. On the Q Exactive (and other instruments with an S-lens), in-source fragmentation using the in-source CID parameter from the DC offset is useful for desolvation and the removal of adducts; however, the larger the fragmentation, the more disruptive the collision. Too much energy can fragment the protein, so the key is finding a balance. Since source fragment CID can be crucial to getting high-quality spectra, Southwick suggests 10–20 volts is a good starting point in optimizing this setting to the protein of interest.
To further illustrate how researchers can optimize the Q Exactive to analyze intact proteins, Southwick compares five case study examples. In case study 1, Southwick begins with a cleaned 17 kDa simple protein. She prepares a 1–2 ml 10 pmol/µl solution in 50% ACN and directly infuses it using a HESI syringe pump at 5–10 µl/min. She points out that if the sample is in limited quantities, direct injection or static nanospray are other options.
Since this protein is less than 25 kDa, it is not difficult to acquire full MS data using a high-resolution scan of 140 K. The small size of the protein also means the scan range of 400–1600 m/z is able to cover the entire charge envelope. Southwick recommends performing three to five microscans to improve the signal, using a capillary temperature of 275°C and an S-lens RF of 60%.
In, case study 2, she attempts to analyze a larger protein at 85 kDa. With this size of protein, the instrument is still able to get full MS data. However, because the size is greater than 50 kDa, the instrument will need to use the lowest resolution, 17.5 K. She also increases the scan range to 80-3,500 m/z. and uses a 60–80% S-lens RF and 20–60 SID(eV).
In case study 3, the protein is 36 kDa, which is in an intermediate range, and therefore there are more options as to what settings are sufficient. Here, a full MS scan at the lowest setting may be enough for relative isoforms and a relative quantification, but for isotopic resolution, SIM or multiplexed SIM (msxSIM) may be needed. The following chart shows the changes in settings for SIM and msxSIM.
|Method type||Full MS||SIM||msxSIM|
|Scan range m/z||700–2,000||850–870||targeted|
|Resolution||17.5||140 K||140 K|
|AGC full MS||3e6||1e6||1e6|
|Max inject time||150 ms||250 ms||250 ms|
In case study 4, Southwick looks at what happens when a sample contains impurities and cannot be directly infused. This situation requires LC. For intact protein analysis, Southwick recommends capillary flow as the best choice because it is robust and allows for lower flow rates that promote sensitivity. Monolith columns can also perform well in intact analysis because the lack of interparticular volume improves mass transfer and separation.
Southwick’s recommended LC method includes a 200 µm x 25 cm ProSwift monolith column (Thermo Scientific). Nano LC is not impossible to do for intact analysis; it is just more difficult, since this method is not as robust and is much less forgiving with sample impurities. For a nano LC analysis, she recommends using either the ProSwift column mentioned above or the 75 µm x 15 cm PLRP-S New Objective column.
Case study 5 deals with complex mixtures containing multiple proteins. For highly complex samples, Southwick recommends fractionating samples based on molecular weight either using size exclusion or GELFrEE fractionation.
For 0–30 kDa samples:
- 140 K resolution
- 4 micro scans
- review data for targeted SIM
For >30 kDa:
- 17.5 resolution
- 5–10 microscans
- review data for targeted SIM
Lastly, Southwick describes some of the unique features of the Q Exactive Plus and the Q Exactive HF instruments that support intact analysis, specifically the protein mode and the enhanced resolution mode. For proteins in the 35–50 kDa range, the protein mode in the Q Exactive Plus mass spectrometer provides less transient decay, improves sensitivity, and enhances resolution. Another advantage of the protein mode is improved signal in heavy chains when analyzing heavy and light chain antibodies. For proteins >50 kDa run analyzed at the lowest resolution, the protein mode and the normal mode do not show significant differences; however, in a protein mixture, protein mode may help boost the signal of proteins >50 kDa.
Enhanced resolution mode (280 K resolution) can work together with protein mode to resolve charge states of proteins (up to 50 kDa). It can also detect ions with higher charge states, giving a richer mass spectrum and a larger protein coverage.
In conclusion, the Q Exactive platform is a valuable asset in intact protein analysis. For further information about other protein discovery applications, check out Planet Orbitrap.