Name: fast enzymatic processing of proteins for ms detection with a flow-through microreactor
Uploaded: 2016-04-06T13:00:00
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 o...

<ol>
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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&#160;</td>
			<td>EMD Millipore</td>
			<td>ZD5311595&#160;</td>
			<td>The MilliQ water system is used to prepare purified DI water</td>
		</tr>
		<tr>
			<td>Pipettor/Eppendorf (1000 &#181;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 &#181;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 &#181;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 &#181;m ID x 360 &#181;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 &#181;m ID x 100 &#181;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 &#181;m ID x 360 &#181;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 &#181;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 mm x 20 cm C18 LC column from Agilent</td>
		</tr>
		<tr>
			<td>Syringe/glass (250 &#181;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&#8221; to 1/32&#8221;)</td>
			<td>Valco</td>
			<td>ZRU1.5FPK</td>
			<td>This union is used to connect the 250 &#181;L syringe to the microreactor for loading the 5 &#181;m particle slurry</td>
		</tr>
		<tr>
			<td>Stainless steel union (1/16&#8221;)</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&#8221;)</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&#8221; 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&#160;</td>
		</tr>
		<tr>
			<td>PEEK tubing (0.015&#8220; ID x 1/16&#8221; 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 &#181;m ID transfer capillary</td>
		</tr>
		<tr>
			<td>PEEK tubing (0.015&#8221; ID x 1/32&#8221; 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&#8221; and 1/32&#8217; 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&#160;</td>
			<td>HP-5183-2069</td>
			<td>The vials are used to prepare sample solutions and the C18 particle slurry&#160;</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 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 (1000 &#181;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 &#181;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 &#181;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&#8221; x 3&#8221;</td>
			<td>These are glass substrates for microchip fabrication</td>
		</tr>
		<tr>
			<td>Male nut fitting (1/16&#8221;)</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></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></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. ...

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