Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

A quick protocol for proteolytic digestion with an in-house built flow-through tryptic microreactor coupled to an electrospray ionization (ESI) mass spectrometer is presented. The fabrication of the microreactor, the experimental setup and the data acquisition process are described.

The overall goal of this protocol is to describe the fabrication of a flow-through tryptic mircoreactor that performs fast enzymatic digestion of proteins for enabling protein identification with an electrospray ionization mass spectrometer. The method can help illustrate the amino acid sequence of isolated and purified proteins to support downstream characterization of structure and function. The main advantage of this protocol is that it is fast, cost-effective, and prone for integration into workflows that make use of microfabricated platforms.

To begin, use a glass capillary cleaver to cut the larger glass capillary tube to a length of seven to eight centimeters, and the smaller one to a length of three to five centimeters. Use a microscope to verify that both capillary ends have a clean, straight cut, without any protruding burrs. Insert the smaller capillary about six millimeters into one end of the larger one.

Then, use a Q-tip to apply a small droplet of glue around the junction of the capillaries, and let the glue cure overnight at room temperature. Next, cut the inserted capillary to a length of about 10 to 15 millimeters. This end will act as the electrospray ionization emitter.

Insert the opposite end of the larger diameter capillary into a 1/32-inch PEEK tube that has been pre-cut between four and five centimeters in length and connected to a PEEK union. Then, insert and tighten the five centimeter-long piece of 1/16 PTFE tubing into the opposite end of the union. In a clean, dry two milliliter glass file, weight out four milligrams of C18 particles and then add 0.5 milliliters of isopropanol.

Close the vial. Place it in ultrasonicator bath and sonicate to disperse the particles evenly in the solution. Next, use a 250 microliter syringe to draw up 200 microliters of the C18 slurry.

Insert the syringe into the 1/16-inch PTFE tubing and dispense the slurry slowly into the large capillary. Observe under the microscope as the capillary fills with particles, until two to three millimeters of particle packing has been achieved. The smaller capillary tube retains these particles in the microreactor through a keystone effect.

Finally, rinse the microreactor with 50 microliters of a 50-to-50 solution of water and acetonitrile, followed by 50 microliters of a solution containing 49 microliters of water, mixed with one microliter of acetonitrile. Disconnect the capillary microreactor from the PEEK union and connect it to a PEEK-T. Then, insert a two centimeter-long platinum wire, insulated with a 1/32-inch PEEK sleeve to provide a leak-free connection into the side arm of the PEEK-T.

Connect a half a meter-long sample transfer capillary to the opposite end of the PEEK-T. Use a 1/32-inch PEEK sleeve to provide a leak-free connection. Secure the PEEK-T on the XYZ stage.

And position the electrospray ionization emitter about two millimeters away from the mass spectrometer inlet capillary. Then, connect the platinum wire to the electrospray ionization power supply source of the mass spectrometer. Also, connect the opposite end of the sample transfer capillary to the stainless steel union, using a 1/16-inch PEEK sleeve to provide a leak-free connection.

Next, connect a four to five centimeter-long piece of 1/16-inch-long PTFE tubing to the opposite end of the stainless steel union. Input the tandem MS data acquisition parameters as described in the accompanying text protocol into the software package that controls the MS instrument. Save them as a method file to ready the setup for performing the analysis.

The LTQ MS system is fitted with a modified ESI source that includes a home-built XYZ stage, which enables the interfacing of mass spectrometer to the various sample input approaches. Load a 250 microliter syringe with an aqueous solution of 50 millimolar bicarbonate dissolved in 2%of acetonitrile. Then, connect the syringe to the microreactor, and place it under the syringe pump.

Rinse the microreactor and two microliters per minute for five minutes, to prepare it for analysis. Then, load the protein sample of interest into a 250 microliter syringe and infuse the sample solution at two microliters per minute for five minutes. Next, place a 250 microliter syringe loaded with a five micromolar trypsin solution under the syringe pump, and infuse over the absorbed protein on the microreactor at two microliters per minute for one to three minutes.

It's critical that one freshly thaws and prepares the trypsin solution before each experiment. This will ensure that the protolytic enzyme retains its activity and its operational pH of the microreactor, which is a pH of about 8. Load another 250 microliter syringe with an aqueous solution consisting of 0%1%of trifluoroacetic acid added to 2%acetonitrile.

Use this solution to quench the proteolytic digestion process by rinsing the microreactor at two microliters per minute for five minutes. Then, switch to a syringe filled with 01%trifluoroacetic acid added to 50%acetonitrile, and turn on the MS Data Acquisition Process in the Electrospray Ionization voltage to about 2000 volts. Use the acidified solution to start eluting the protein digest from the microreactor at 300 nanoliters per minute for 20 to 30 minutes.

Acquire mass spec data using the data acquisition parameters provided in the accompanying text protocol. Process the LTQ MS raw files using a minimally redundant protein database by first uploading the raw data in the search engine. Then, set the parent and fragment ion mass tolerances to two and one daltons respectively, and use only fully tryptic fragments with up to two missed cleavages for the search.

In addition, set the high and medium confidence false discovery rates to 1%and 3%respectively, and do not allow for post-translational modifications, unless specifically looking for particular modifications. Finally, filter the data to select only the high confidence peptide matches. Shown here is a mass spectrum comprised of tryptic peptides that were generated from a protein mixture that was subjected to proteolysis in the microreactor.

These labels represent the most intense tryptic peptide ions and provide their sequence and corresponding protein identifier. This microreactor provides an easy-to-implement experiment set up for performing enzymatic digestion of proteins and enables a mass analysis and identification in less than 30 minutes. While attempting this procedure, it is important to remember that preserving the cleanliness of the vials and instrumentation that come into contact with solutions is critical to avoiding sample contamination.

Following this procedure, other mass spectrometry data acquisition methods can be used to enable a better characterization of the generative peptides and answer additional questions related to structure of proteins. After its development, this technique paved the way for researchers in the field of proteonomics to develop faster protocols for the analysis of proteins and explore the development of novel microphalytic platforms that enable the high throughput exploration of biological samples. This technique is limited to the analysis of rather simple protein mixtures that generate 25 to 50 peptides.

These peptides can be analyzed by simple infusion experiments and do not necessitate liquid chromatographics separation prior to MS analysis. Don't forget that working with organic solvents can be extremely hazardous, and precautions such as avoiding open flames should always be taken while performing this procedure.