Name: fast enzymatic processing of proteins for ms detection with a flow-through microreactor
Uploaded: 2016-04-06T13:00:00.000+00:00
Duration: 9 min 49 s
Description: Watch this Scientific Journal Video about Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor at JoVE.com

This work was supported by NSF/DBI-1255991 grant to IML.

1. Preparation of the Capillary Microreactor
<ol>
	<li>Cut the 100 &#181;m internal diameter (ID) x 360 &#181;m outer diameter (OD) capillary to a length of 7-8 cm, and the 20 &#181;m ID x 90 &#181;m OD capillary to 3-5 cm with the glass capillary cleaver; verify under the microscope that both capillary ends have a clean, straight cut, without any protruding burrs.</li>
	<li>Insert the 20 &#181;m ID x 90 &#181;m OD capillary into one end of the 100 &#181;m ID x 360 &#181;m OD capillary, for a length of ~6 mm; in case of need, observe this operation under a microscope.</li>
	<li>Apply a small droplet of glue E6000 around the junction of the 90 &#181;m OD/100 &#181;m ID capillaries with the end of a Q-tip, and let the glue cure O/N at RT.</li>
	<li>Cut the inserted 20 &#181;m ID x 90 &#181;m OD capillary to a length of ~10-15 mm; this will be the electrospray ionization emitter.</li>
	<li>Pre-cut the 1/32&#34; OD PEEK, 1/16&#34; OD PEEK and 1/16&#34; OD PTFE tubing in pieces of 4-5 cm in length; these will be the sleeves used in capillary unions for providing a leak-free connection.</li>
	<li>Connect the opposite end of the 100 &#181;m ID x 360 &#181;m OD capillary to a PEEK union; use a 1/32&#34; PEEK sleeve for providing a leak-free connection.</li>
	<li>Insert/tighten a piece of ~5 cm long 1/16&#34; PTFE tubing into the opposite end of the union.</li>
	<li>Weigh ~4 mg of C18 (5 &#181;m) particles in a pre-cleaned/dried 2 ml glass vial; add 0.5 ml isopropanol, close the vial and disperse the slurry in the ultrasonicator bath.</li>
	<li>Withdraw with a 250 &#181;l syringe approximately 200 &#181;l slurry, insert the syringe needle in the 1/16&#34; PTFE tubing of the PEEK union, and dispense slowly the slurry into the 100 &#181;m ID x 360 &#181;m OD capillary; observe under the microscope as the capillary fills with particles until a length of 2-3 mm packing is achieved; this will be the microreactor (Figure 1). The 20 &#181;m ID capillary retains the particles in the microreactor through a Keystone effect.15</li>
	<li>Rinse the microreactor with ~50 &#181;l solution of H2O/CH3CN 50:50 v/v, and then with ~50 &#181;l solution of H2O/CH3CN 98:2 v/v.</li>
</ol>
2. Preparation of Sample Solutions
Note: Operations that involve handling of organic solvents and acids and solution preparation are to be performed in a fume hood. Wear goggles, gloves and protective clothing.
<ol>
	<li>Rinse with CH3OH and dry a few glass vials (4 ml) and polypropylene tubes (15 ml).</li>
	<li>Prepare 10 ml of a solution of H2O/CH3CN (98:2 v/v) in a 15 ml polypropylene tube by mixing 9.8 ml H2O with 200 &#181;l CH3CN; mix by sonication.</li>
	<li>Prepare 10 ml of a solution of H2O/CH3CN (50:50 v/v) in a 15 ml polypropylene tube by mixing 5 ml H2O with 5 ml CH3CN; mix by sonication.</li>
	<li>Prepare 4 ml of an acidified aqueous solution (TFA 0.01% v/v) by adding 4 &#181;l TFA(10%) to 4 ml H2O/CH3CN (98:2 v/v) in a 4 ml glass vial; mix by sonication.</li>
	<li>Prepare 4 ml of an acidified organic solution (TFA 0.01% v/v) by adding 4 &#181;l TFA(10%) to 4 ml H2O/CH3CN (50:50 v/v) in a 4 ml glass vial; mix by sonication.</li>
	<li>Weigh 16 mg NH4HCO3 with an analytical microbalance in a 4 ml glass vial; add 4 ml of the H2O/CH3CN (98:2 v/v) solution and disperse by sonication; this results in a 50 mM solution of NH4HCO3 (pH~7.8) in aqueous buffer.</li>
	<li>Weigh 5 mg of a standard protein (e.g., bovine serum albumin, hemoglobin &#945;/&#946;, cytochrome C, carbonic anhydrase, &#945;-2-HS-glycoprotein, etc.) with an analytical microbalance in a 4 ml glass vial; add 4 ml DI water and disperse by sonication; this typically results in a high &#181;M-level concentration solution.</li>
	<li>Prepare 1 ml protein solution (1-2 &#181;M) by diluting the high &#181;M-level concentration solution with an appropriate volume of NH4HCO3 (50 mM) aqueous buffer; mix by sonication.</li>
	<li>Prepare the trypsin solution (5 &#181;M) by dissolving 20 &#181;g sequencing-grade trypsin in 170 &#181;l NH4HCO3 (50 mM) aqueous buffer; disperse by sonication.</li>
</ol>
3. Experimental Setup&#160;
Note: The LTQ-MS system is fitted with a modified ESI source that includes a home-built XYZ-stage which enables interfacing of the mass spectrometer to various sample input approaches.
<ol>
	<li>Disconnect the capillary microreactor from the PEEK union and connect to the PEEK Tee.</li>
	<li>Insert a ~2 cm long Pt wire in the side-arm of the PEEK Tee; use a 1/32&#34; PEEK sleeve for providing a leak-free connection and for insulating the Pt wire.</li>
	<li>Connect a 50 &#181;m ID x 360 &#181;m OD (~0.5 m long) sample transfer capillary to the opposite end of the PEEK Tee; use a 1/32&#34; PEEK sleeve for providing a leak-free connection.</li>
	<li>Secure the PEEK Tee on the XYZ-stage and position the ESI emitter ~2 mm away from the mass spectrometer inlet capillary.</li>
	<li>Connect the Pt wire to the ESI power supply source of the mass spectrometer.</li>
	<li>Connect the opposite end of the sample transfer capillary to the stainless steel union; use a 1/16&#34; PEEK sleeve for providing a leak-free connection.</li>
	<li>Connect a 4-5 cm long 1/16&#34; PTFE tubing to the opposite end of the stainless steel union and insert the 250 &#181;l syringe needle in the PTFE tubing; turn on the pump and set the flow rate at the desired value (e.g., 2 &#181;l/min).</li>
	<li>Input tandem MS data acquisition parameters into the software package that controls the MS instrument and save as a method file to ready the setup for performing the analysis; for the LTQ-MS system, use the following data dependent analysis parameters: dynamic exclusion enabled for 180 sec with exclusion mass width &#177;1.5 m/z; one MS scan followed by one zoom and MS2 scans on the top 5 most intense peaks, zoom scan width &#177;5 m/z; collision induced dissociation (CID) parameters set at isolation width 3 m/z, normalized collision energy 35%, activation Q 0.25, and activation time 30 msec.</li>
</ol>
4. Microfluidic Setup&#160;
<ol>
	<li>Prepare a microfluidic device from glass or polymeric substrates;16-18 the layout of the device is provided in Figure 2A, consisting of a microreactor (1) (0.13 mm width x 2 mm length x 50 &#181;m deep) connected to an inlet (2), outlet (3) and a side port (4); the interconnecting channels for fluidic manipulations are ~110 &#181;m wide and ~50 &#181;m deep; a multichannel structure (5), consisting of ~1.5-2 &#181;m deep x 10 &#956;m wide x 100 &#956;m long microchannels placed 25 &#181;m apart, will act as a filter for retaining the particles in the microreactor.</li>
	<li>Enable fluid-delivery connections to the chip by gluing specialized connectors to the inlet ports, or by devising a polymeric stand that accommodates fittings for fluid delivery (Figure 2B).16</li>
	<li>Prepare a slurry of C18 particles (5 &#181;m) as described in step 1.8; deliver the slurry to the microreactor with a syringe by using 1/16&#34; PTFE tubing connected to the chip side port (4); seal the port after particle loading with epoxy glue and cure for 1 hr in an oven at 90 &#186;C.16</li>
	<li>Insert a capillary (20 &#181;m ID x 90 &#181;m OD x 10 mm length) in the outlet port, seal with E6000 glue, and let cure O/N; this will become the ESI emitter (6).</li>
	<li>Connect a 50 &#181;m ID x 360 &#181;m OD (~0.5 m long) sample transfer capillary to the chip inlet port (2); connect the opposite end of the capillary to a 250 &#181;l syringe and the syringe pump, as described in 3.6-3.7.</li>
	<li>Place a light-weight PEEK Tee connector on the sample transfer line just prior to the inlet port (2) and insert a Pt wire on the side-arm of the Tee, as described in 3.2; this will be the ESI electrode.</li>
	<li>Secure the microfluidic device on the XYZ stage with the ESI emitter positioned ~2 mm away from the mass spectrometer inlet capillary; prepare the MS for analysis as described in 3.8.</li>
</ol>
5. Sample Loading, Proteolytic Digestion and Elution for MS Analysis
Note: All solution/sample transfer steps are performed with the aid of a syringe pump and should allow for a few additional minutes to complete, to compensate for the dead-volumes associated with the transfer lines to and from the microreactor; the necessary time will depend on the flow rates involved.
<ol>
	<li>Rinse the microreactor and prepare it for analysis by infusing an aqueous solution of NH4HCO3 (50 mM) in H2O/CH3CN (98:2 v/v) at 2 &#181;l/min for 5 min; use a 250 &#181;l syringe.</li>
	<li>Load the protein sample (1 &#181;M) on the microreactor by infusing the sample solution at 2 &#181;l/min for 5 min; use a 250 &#181;l syringe.</li>
	<li>Infuse the trypsin solution (5 &#181;M) over the adsorbed protein on the microreactor at 2 &#181;l/min for 1-3 min.</li>
	<li>Quench the proteolytic digestion process by rinsing the microreactor with an acidified aqueous solution of TFA (0.01% v/v) in H2O/CH3CN (98:2 v/v) at 2 &#181;l/min for 5 min; use a 250 &#181;l syringe.</li>
	<li>Start eluting the protein digest from the microreactor by infusing an acidified organic solution of TFA (0.01% v/v) in H2O/CH3CN (50:50 v/v) at 300 nl/min.</li>
	<li>Turn on the MS data acquisition process and the ESI voltage (~2,000 V); acquire MS data using the data acquisition parameters provided in step 3.8; observe the elution of the tryptic peptides.</li>
</ol>
6. Data Processing
<ol>
	<li>Process the tandem MS data with a search engine compatible with the raw MS data format.
	<ol>
		<li>Process the LTQ-MS raw files using an organism specific minimally redundant protein database; upload the raw data in the search engine, set the parent and fragment ion mass tolerances to 2 and 1 Da, respectively, and use only fully tryptic fragments with up to two missed cleavages for the search; do not allow for posttranslational modifications; set the high and medium-confidence false discovery rates (FDR) to 1% and 3%, respectively, and do not allow for posttranslational modifications.</li>
	</ol>
	</li>
	<li>Filter the data to select only high-confidence peptide matches to the proteins in the database; evaluate the protein and peptide-level results.</li>
</ol>

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The authors declare no competing financial interests.

The microreactor described in this work provides an easy-to-implement experimental setup for performing enzymatic digestion of proteins to enable MS analysis and identification in less than 30 min. The distinct advantages of this system, in comparison to conventional approaches, include simplicity, speed, low reagent consumption and low costs. In particular, there is no need for expensive immobilized trypsin beads and cartridges. The preparation of the capillary microreactor is straightforward (Figure 1A...

The identification and characterization of purified proteins is frequently achieved by using MS techniques. The protein is digested with an enzyme and its peptides are further analyzed by MS by using a simple infusion experimental setup. Proteolytic digestion is necessary for generating small peptide fragments that fall in the useful mass range of most MS analyzers, and that can be easily fragmented through low energy collision induced dissociation to generate amino acid sequence information. For isolated proteins or simple protein mixtures, there is no further need for chromatographic separation of peptides prior to MS detection. A mixture of 25-50 peptides can be easily analyzed by infusing the sample with a syringe pump directly in the MS ion source.
The mass spectrometer can perform the analysis and confirm the sequence of a protein within a short time-frame. With modern data acquisition methods, this process can be accomplished within a few minutes or even seconds. The limiting factor in completing the entire process on a short time-scale is the proteolytic digestion step. Typically, this is performed over a few hours (or O/N), in solution, at 37 &#186;C, using substrate:enzyme ratios of (50-100):1. To reduce the enzymatic digestion time to minutes or seconds, immobilized enzyme microreactors, in the form of microfluidic reactors or commercially available cartridges, have been described.1-6 Typically, the enzyme is immobilized by covalent, non-covalent/physical adsorption, complex formation or encapsulation,3,6 the enhanced efficiency of the enzymatic process being enabled by the large surface-to-volume and enzyme-to-substrate ratios. Additional advantages of immobilized reactors include reduced autolysis and interference from the enzyme in MS analysis, improved enzyme stability and reusability. A variety of approaches, using glass or polymeric microfabricated devices have been described, using enzymes immobilized on magnetic beads by antibody-antigen interactions,7,8 entrapped in gold nanoparticle networks,9 encapsulated in titania-alumina sol-gels10 and nanozeolites,11 or captured through Ni-NTA or His-Tag complex formation.6 Alternatively, open-tubular capillaries with immobilized enzymes have been developed, as well.12 Moreover, enhanced proteolytic cleavage has been demonstrated by using controlled microwave irradiation13 or pressure-assisted or pressure cycling technology (PCT) for reducing the reaction times to 30-120 min.14
Despite the multiple advantages of immobilized enzyme reactors, the costs of commercial cartridges is high, the availability of microfluidic devices for routine use is limited, and the use of microwave or PCT technologies results in need for additional instrumentation. The goal of this work was to develop a method that circumvents these disadvantages, and that can be easily implemented in every laboratory to empower researchers with a simple and effective approach for performing enzymatic cleavage of proteins in preparation for MS analysis within minutes. The approach relies on the use of hydrophobic, C18-particles which are pre-loaded in a capillary or microfluidic device, and the adsorption of the protein(s) of interest on these particles followed by enzymatic digestion during the infusion of the enzyme over the packed bed and captured protein(s). In this approach, the substrate is immobilized through non-covalent interactions, and the enzyme is infused over the immobilized protein. The proteolytic digestion efficiency is increased by the large particle surface areas that expose the protein for enzymatic processing, reduced distances and diffusion times to and from the surface of particles, improved mass transfer, no covalent attachment that may affect the activity of the enzyme, ability to quickly evaluate combinations of different enzymes, disposability, and multiplexing if the process is executed in a microfluidic format. This approach is demonstrated with the use of a mixture of standard proteins and trypsin-the most commonly used enzyme for proteolytic digestion prior to ESI-MS detection. The mass spectrometer used for detection in this study was a linear trap quadrupole (LTQ) instrument.

<table><tbody><tr><td>Ion trap ESI-MS</td><td>Thermo Electron</td><td>LTQ</td><td>The LTQ mass spectrometer is used for acquiring tandem MS data</td></tr><tr><td>XYZ stage</td><td>Newport</td><td>Multiple parts</td><td>The home-built XYZ stage is used to adapt the commercial LTQ nano-ESI source to receive input from various sample delivery systems</td></tr><tr><td>Stereo microscope</td><td>Edmund optics</td><td>G81-278</td><td>The microscope is used to observe the microreactor packing process</td></tr><tr><td>Analytical balance/Metler</td><td>VWR</td><td>46600-204</td><td>The balance is used to weigh the protein samples</td></tr><tr><td>Ultrasonic bath/Branson</td><td>VWR</td><td>33995-540</td><td>The sonic bath is used for mixing/homogenizing the samples and dispersing the C18 particle slurry</td></tr><tr><td>Syringe pump 22</td><td>Harvard Apparatus</td><td>552222</td><td>The micropump is used for loading, rinsing and eluting the sample and the enzyme on and from the packed capillary microreactor</td></tr><tr><td>Milli-Q ultrapure water system</td><td>EMD Millipore</td><td>ZD5311595&amp;nbsp;</td><td>The MilliQ water system is used to prepare purified DI water</td></tr><tr><td>Pipettor/Eppendorf (1,000&amp;nbsp;&amp;micro;l)</td><td>VWR</td><td>53513-410</td><td>The pipettor is used to measure small volumes of sample solutions</td></tr><tr><td>Pipettor/Eppendorf (100&amp;nbsp;&amp;micro;l)</td><td>VWR</td><td>53513-406</td><td>The pipettor is used to measure small volumes of sample solutions</td></tr><tr><td>Pipettor/Eppendorf (10&amp;nbsp;&amp;micro;l)</td><td>VWR</td><td>53513-402</td><td>The pipettor is used to measure small volumes of sample solutions</td></tr><tr><td>Fused silica capillary (100 &amp;micro;m ID x 360 &amp;micro;m OD)</td><td>Polymicro Technologies</td><td>TSP100375</td><td>This capillary is used for the fabrication of the microreactor</td></tr><tr><td>Fused silica capillary (20&amp;nbsp;&amp;micro;m ID x 100 &amp;micro;m OD)</td><td>Polymicro Technologies</td><td>TSP020090</td><td>This capillary is used for the fabrication of the ESI emitter</td></tr><tr><td>Fused silica capillary (50&amp;nbsp;&amp;micro;m ID x 360 &amp;micro;m OD)</td><td>Polymicro Technologies</td><td>TSP050375</td><td>This capillary is used to transfer the samples and the eluent from the syringe pump to the capillary microreactor</td></tr><tr><td>Glass capillary cleaver</td><td>Supelco</td><td>23740-U</td><td>This is a tool for cutting fused silica capillaries at the desired length</td></tr><tr><td>Glue</td><td>Eclectic Products</td><td>E6000 Craft</td><td>This glue is used for securing the ESI emitter into the capillary microreactor or the microfluidic chip</td></tr><tr><td>Epoxy glue</td><td>Epo-Tek</td><td>353NDT</td><td>This glue is used to seal the microfluidic inlet hole through which the C18 particles are loaded</td></tr><tr><td>Reversed phase C18 particles (5&amp;nbsp;&amp;micro;m)</td><td>Agilent Technologies</td><td>Zorbax 300SB-C18</td><td>These are C18 particles on which the proteins are adsorbed; the particles were extracted from a 4&amp;nbsp;mm x 20 cm C18 LC column from Agilent</td></tr><tr><td>Syringe/glass (250 &amp;micro;l)</td><td>Hamilton</td><td>81130-1725RN</td><td>The glass syringes are used to load the C18 particle slurry in the capillary microreactor and to deliver the sample and eluents to the microreactor</td></tr><tr><td>Internal reducing PEEK Union (1/16&amp;rdquo; to 1/32&amp;rdquo;)</td><td>Valco</td><td>ZRU1.5FPK</td><td>This union is used to connect the 250 &amp;micro;l syringe to the microreactor for loading the 5 &amp;micro;m particle slurry</td></tr><tr><td>Stainless steel union (1/16&amp;rdquo;)</td><td>Valco</td><td>ZU1XC</td><td>The stainless steel union is used to connect the glass syringe needle to the infusion capillary</td></tr><tr><td>Microvolume PEEK Tee connector (1/32&amp;rdquo;)</td><td>Valco</td><td>MT.5XCPK</td><td>The Peek tee is used to connect the sample transfer capillary to the capillary microreactor; on its side arm, it enables the insertion of the Pt wire</td></tr><tr><td>Tee connector (light weight)</td><td>Valco</td><td>C-NTXFPK</td><td>This Tee connector is used to apply ESI voltage to the microfluidic chip through the sample transfer line</td></tr><tr><td>Pt wire (0.404 mm)</td><td>VWR</td><td>66260-126</td><td>The Pt wire provides electrical connection for ESI generation and is connected to the mass spectrometer ESI power supply</td></tr><tr><td>PTFE tubing (1/16&amp;rdquo; OD)</td><td>Valco</td><td>TTF115-10FT</td><td>The Teflon tubing is used to enable an air-tight connection between the syringe needle and the stainless steel union&amp;nbsp;</td></tr><tr><td>PEEK tubing (0.015&amp;rdquo; ID x 1/16&amp;rdquo; OD)</td><td>Upchurch Scientific</td><td>1565</td><td>The Peek tubing is used as a sleeve to enable an air-tight connection between the stainless steel union and the 50&amp;nbsp;&amp;micro;m ID transfer capillary</td></tr><tr><td>PEEK tubing (0.015&amp;rdquo; ID x 1/32&amp;rdquo; OD)</td><td>Valco</td><td>TPK.515-25</td><td>The Peek tubing is used as a sleeve to enable a leak-free connection between the fused silica capillaries and the Peek Tee</td></tr><tr><td>Clean-cut polymer tubing cutter</td><td>Valco</td><td>JR-797</td><td>This cutter is used to pre-cut the 1/16&amp;rdquo; and 1/32&amp;rsquo; Peek polymer tubing that is used as sleeve for leak-free connections in pieces of ~4-5 cm in length</td></tr><tr><td>Amber vial (2 ml)</td><td>Agilent&amp;nbsp;</td><td>HP-5183-2069</td><td>The vials are used to prepare sample solutions and the C18 particle slurry&amp;nbsp;</td></tr><tr><td>Amber vial (4 ml)</td><td>VWR</td><td>66011-948</td><td>The vials are used to prepare sample solutions</td></tr><tr><td>Polypropylene tube (15&amp;nbsp;ml)</td><td>Fisher</td><td>12-565-286D</td><td>The vials are used to prepare buffer solutions</td></tr><tr><td>Cylinder (100 ml)</td><td>VWR</td><td>24710-463</td><td>The cylinder is used to measure volumes of solvent</td></tr><tr><td>Cylinder (10 ml)</td><td>VWR</td><td>24710-441</td><td>The cylinder is used to measure volumes of solvent</td></tr><tr><td>Pipette tips (1,000 &amp;micro;l)</td><td>VWR</td><td>83007-386</td><td>The pipette tips are used to measure small volumes of sample solutions</td></tr><tr><td>Pipette tips (100 &amp;micro;l)</td><td>VWR</td><td>53503-781</td><td>The pipette tips are used to measure small volumes of sample solutions</td></tr><tr><td>Pipette tips (10 &amp;micro;l)</td><td>VWR</td><td>53511-681</td><td>The pipette tips are used to measure small volumes of sample solutions</td></tr><tr><td>Glass substrates</td><td>Nanofilm</td><td>B270 white crown, 3&amp;rdquo; x 3&amp;rdquo;</td><td>These are glass substrates for microchip fabrication</td></tr><tr><td>Male nut fitting (1/16&amp;rdquo;)</td><td>Upchurch</td><td>P203X</td><td>This fitting is used for connecting transfer capillaries to the microfluidic chip</td></tr><tr><td>Nanoport assembly</td><td>Upchurch</td><td>N-122H</td><td>This fitting is used for connecting transfer capillaries to the microfluidic chip</td></tr><tr><td>Protein standards</td><td>Sigma</td><td>Multiple #</td><td></td></tr><tr><td>Acetonitrile, HPLC grade</td><td>Fisher</td><td>A955</td><td></td></tr><tr><td>Methanol, HPLC grade</td><td>Fisher</td><td>A452</td><td></td></tr><tr><td>Isopropanol, HPLC grade</td><td>Sigma</td><td>650447</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>Sigma</td><td>302031</td><td></td></tr><tr><td>Ammonium bicarbonate</td><td>Aldrich</td><td>A6141</td><td></td></tr><tr><td>Trypsin, sequencing grade</td><td>Promega</td><td>V5111</td><td></td></tr></tbody></table>

fast enzymatic processing of proteins for ms detection with a flow-through microreactor

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. This step is necessary for the generation of small molecular weight peptides, generally with MW &#60; 3,000-4,000 Da, that fall within the effective scan range of mass spectrometry instrumentation. Conventional protocols involve O/N enzymatic digestion at 37 &#186;C. Recent advances have led to the development of a variety of strategies, typically involving the use of a microreactor with immobilized enzymes or of a range of complementary physical processes that reduce the time necessary for proteolytic digestion to a few minutes (e.g., microwave or high-pressure). In this work, we describe a simple and cost-effective approach that can be implemented in any laboratory for achieving fast enzymatic digestion of a protein. The protein (or protein mixture) is adsorbed on C18-bonded reversed-phase high performance liquid chromatography (HPLC) silica particles preloaded in a capillary column, and trypsin in aqueous buffer is infused over the particles for a short period of time. To enable on-line MS detection, the tryptic peptides are eluted with a solvent system with increased organic content directly in the MS ion source. This approach avoids the use of high-priced immobilized enzyme particles and does not necessitate any aid for completing the process. Protein digestion and complete sample analysis can be accomplished in less than ~3 min and ~30 min, respectively.

A representative result of a proteolytic digestion process performed simultaneously on a mixture of proteins, with the above-described microreactors (Figures 1 or 2), is provided in Table 1. The table comprises the unique peptide sequences that identify a particular protein, the cross correlation score (Xcorr) (i.e., a score that characterizes the quality of the experimental-to-theoretical match for the corresponding tandem mass spectrum), the nu...

Watch this Scientific Journal Video about Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor at JoVE.com

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 vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. ...

fast-enzymatic-processing-proteins-for-ms-detection-with-flow-through

Research

JoVE Journal

Bioengineering

7.9K Views.  Virginia Tech. 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.

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

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Biochemistry

A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, glass coverslips are functionalized in a one-step reaction with silane-PEG-biotin. Flow cells are constructed by sandwiching an adhesive tape with pre-cut channels between a functionalized coverslip and a PDMS slab containing inlet and outlet holes. Multiple channels are integrated into one flow cell and the flow of reagents into each channel can be fully automated, which significantly increases the assay throughput and reduces hands-on time per assay. Inside each channel, biotin-&#955;-DNAs are immobilized on the surface and a laminar flow is applied to flow-stretch the DNAs. The DNA molecules are stretched to &#62;80% of their contour length and serve as spatially extended templates for studying the binding and transport activity of fluorescently labeled molecules. The trajectories of single molecules are tracked by time-lapse Total Internal Reflection Fluorescence (TIRF) imaging. Raw images are analyzed using streamlined custom single particle tracking software to automatically identify trajectories of single molecules diffusing along DNA and estimate their 1D diffusion constants.

This protocol demonstrates a simple, robust and high throughput single molecule flow-stretching assay for studying one-dimensional (1D) diffusion of molecules along DNA.

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, ...

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Platform Incubator with Movable XY Stage: A New Platform for Implementing In-Cell Fast Photochemical Oxidation of Proteins

Fast Photochemical Oxidation of proteins (FPOP) coupled with mass spectrometry (MS) has become an invaluable tool in structural proteomics to interrogate protein interactions, structure, and protein conformational dynamics as a function of solvent accessibility. In recent years, the scope of FPOP, a hydroxyl radical protein foot printing (HRPF) technique, has been expanded to protein labeling in live cell cultures, providing the means to study protein interactions in the convoluted cellular environment. In-cell protein modifications can provide insight into ligand induced structural changes or conformational changes accompanying protein complex formation, all within the cellular context. Protein footprinting has been accomplished employing a customary flow-based system and a 248 nm KrF excimer laser to yield hydroxyl radicals via photolysis of hydrogen peroxide, requiring 20 minutes of analysis for one cell sample.To facilitate time-resolved FPOP experiments, the use of a new 6-well plate-based IC-FPOP platform was pioneered. In the current system, a single laser pulse irradiates one entire well, which truncates the FPOP experimental time frame resulting in 20 seconds of analysis time, a 60-fold decrease. This greatly reduced analysis time makes it possible to research cellular mechanisms such as biochemical signaling cascades, protein folding, and differential experiments (i.e., drug-free vs. drug bound) in a time-dependent manner. This new instrumentation, entitled Platform Incubator with Movable XY Stage (PIXY), allows the user to perform cell culture and IC-FPOP directly on the optical bench using a platform incubator with temperature, CO2 and humidity control. The platform also includes a positioning stage, peristaltic pumps, and mirror optics for laser beam guidance. IC-FPOP conditions such as optics configuration, flow rates, transient transfections, and H2O2 concentration in PIXY have been optimized and peer-reviewed. Automation of all components of the system will reduce human manipulation and increase throughput.

A new static platform is used to characterize protein structure and interaction sites in the native cell environment utilizing a protein footprinting technique called in-cell fast photochemical oxidation of proteins (IC-FPOP).

Fast Photochemical Oxidation of proteins (FPOP) coupled with mass spectrometry (MS) has become an invaluable tool in structural proteomics to interrogate ...

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

Biology

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits the dielectric properties of thin insulator films to enhance the charge density and hence boost the electrowetting effect. The presence of charges results in an electrically induced spreading of the droplet which permits purposeful manipulation across a hydrophobic surface. Here, we demonstrate EWOD-based protocol for sample processing and detection of four categories of antigens, using an automated surface actuation platform, via two variations of an Enzyme-Linked Immunosorbent Assay (ELISA) methods. The ELISA is performed on magnetic beads with immobilized primary antibodies which can be selected to target a specific antigen. An antibody conjugated to HRP binds to the antigen and is mixed with H2O2/Luminol for quantification of the captured pathogens. Assay completion times of between 6 and 10 min were achieved, whilst minuscule volumes of reagents were utilized.

Electrowetting-based digital microfluidic is a technique that utilizes a voltage-driven change in the apparent contact angle of a microliter-volume droplet to facilitate its manipulation. Combining this with functionalized magnetic beads enables the integration of multiple laboratory unit operations for sample preparation and identification of pathogens using Enzyme-linked Immunosorbent Assay (ELISA).

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits ...

Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics Under Physiological Conditions

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks of Parkinson's disease. Yet, much of its biological activity, as well as its aggregation, centrally involves the soluble monomer form of the protein. Elucidation of the molecular mechanisms of aSyn biology and pathophysiology requires structurally highly resolved methods and is sensitive to biological conditions. Its natively unfolded, meta-stable structures make monomeric aSyn intractable to many structural biology techniques. Here, the application of one such approach is described: hydrogen/deuterium-exchange mass spectrometry (HDX-MS) on the millisecond timescale for the study of proteins with low thermodynamic stability and weak protection factors, such as aSyn. At the millisecond timescale, HDX-MS data contain information on the solvent accessibility and hydrogen-bonded structure of aSyn, which are lost at longer labeling times, ultimately yielding structural resolution up to the amino acid level. Therefore, HDX-MS can provide information at high structural and temporal resolutions on conformational dynamics and thermodynamics, intra- and inter-molecular interactions, and the structural impact of mutations or alterations to environmental conditions. While broadly applicable, it is demonstrated how to acquire, analyze, and interpret millisecond HDX-MS measurements in monomeric aSyn.

The structural ensemble of monomeric alpha-synuclein affects its physiological function and physicochemical properties. The present protocol describes how to perform millisecond hydrogen/deuterium-exchange mass spectrometry and subsequent data analyses to determine conformational information on the monomer of this intrinsically disordered protein under physiological conditions.

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks ...