This work was supported by the Clinical Proteomic Technologies for Cancer initiative, 5U24CA126477-04.

1) Prepare lectin-conjugated POROS columns
<ol>
<li>Put on a mask to protect against inhalation of POROS-AL beads during steps 1.1 - 1.2. Weigh out the desired amount of POROS beads (100 mg beads/300 &mu;l final volume) and transfer into a clean eppendorf tube.</li>
<li>Wash beads with addition of 1 mL phosphate-buffered saline (PBS). Pellet beads by centrifugation in a microcentrifuge at maximum speed for 3 minutes. Remove supernatant and repeat the wash.</li>
<li>Weigh out the desired amount of unconjugated lectin (1 - 4 mg/ 200 &mu;l beads) and transfer to a clean eppendorf tube. Add PBS to form a 5 - 20 mg/mL solution. Reserve 25 &mu;l of this solution.</li>
<li>Transfer the remaining lectin solution to the POROS beads. Add sodium cyanoborohydride to a final concentration of 50 mM. Place the tube on a rocker and react overnight at room temperature. (Note: sodium cyanoborohydride is toxic and must be handled in a fume hood. Contaminated waste must be disposed of appropriately.)</li>
<li>Pellet the POROS beads as in step 1.2. Remove supernatant and save as the post-conjugation solution.</li>
<li>Wash beads with 1 mL Quenching Buffer (1 M Tris-Cl, pH 7.4). Pellet beads as in step 1.2 and safely discard supernatant.</li>
<li>Block the remaining reactive sites on the POROS beads with 1 mL Quenching Buffer. Add sodium cyanoborohydride to a final concentration of 50 mM. Place the tube on a rocker and incubate at room temperature for 30 min.</li>
<li>Pellet beads as in step 1.2 and discard supernatant.</li>
<li>Wash beads with 1 mL 1 M NaCl. Pellet beads and discard supernatant. Repeat four times for a total of five washes. The beads are now ready to pack.</li>
<li>To determine the amount of protein that was conjugated to the POROS beads, perform a protein concentration assay on the pre-conjugated and post-conjugated lectin solutions. The difference in concentrations is the amount of protein that was conjugated to the beads. The amount of protein conjugated per bead volume equals the concentration of lectin on the beads. Target lectin concentrations are between 2 -20 mg/mL.</li>
</ol>
2) Packing the lectin-conjugated POROS beads into a PEEK column
<ol>
<li>Assemble the packing system (description follows) and support it on a metal ring stand. The packing system consists of, from bottom to top, pressure restrictor, end column coupler 1, frit, column (2 x 50 mm), end column coupler 2, column connector, end column coupler 3, column (4.5 x 50 mm), end column coupler 4 and end fitting. The upper column serves as a reservoir for the packing material.</li>
<li>Resuspend the POROS beads in desired volume of Buffer A (10 mM Tris buffer, pH 7.4, 150 mM sodium chloride, 10 mM calcium chloride, 10 mM magnesium chloride). If packing one column (~200 &mu;l bed volume), resuspend in 400 &mu;l Buffer A.</li>
<li>Transfer conjugated POROS beads into the upper column (reservoir). Add Buffer A to the reservoir until the buffer reaches the top of the column. Gently place the end fitting onto the top of the column, trying to avoid air bubbles in the column. Connect the end fitting of the upper column to the HPLC system.</li>
<li>Pack the column by flowing Buffer A through the system. Start with a flow rate of 0.5 mL/min. Increase the flow rate by 0.5 mL/min each minute until either 4 mL/min or the maximum pressure (3000 psi) has been reached. Continue at the maximum possible flow rate until at least 35 mL of Buffer A have passed through the column.</li>
<li>Turn off the pumps and allow the pressure on the column to drop to &lt; 20 psi.</li>
<li>Gently, disassemble the packing system, starting from the top. When the end column coupler 2 is reached, remove carefully. Some packed material should be extruding from the top of the column. With a razor blade or similar sharp edge, gently wipe away the extruding beads, leaving a bead surface that is even with the top of the column. Do not apply pressure to the beads while doing this.</li>
<li>Disengage the packed column from the ring stand. Place a new frit into a new end coupler and turn the packed column over to connect this with the frit/end coupler on the open (formerly top) column end.</li>
<li>Label the column appropriately. It is now ready for use. When not in use, the column may be stored in PBS with 0.02% sodium azide at 4 &deg;C for up to 6 months.</li>
</ol>
3) Program the HPLC method
<ol>
<li>The details of programming your HPLC will vary according to the specifics of the manufacturer's software. We use a Michrom Paradigm MG4 HPLC. On this machine, methods are built and accessed under the "Setup" tab at the top of the screen. Program the following method: 
<table border="1" cellspacing="0" cellpadding="0">
<tbody>
 <tr>
 <td>Time</td>
 <td>Flow rate (&mu;l/min)</td>
 <td>% A</td>
 <td>% B</td>
 </tr>
 <tr>
 <td>0:00</td>
 <td>50</td>
 <td>100</td>
 <td>0</td>
 </tr>
 <tr>
 <td>9:00</td>
 <td>50</td>
 <td>100</td>
 <td>0</td>
 </tr>
 <tr>
 <td>9:01</td>
 <td>500</td>
 <td>0</td>
 <td>100</td>
 </tr>
 <tr>
 <td>13:50</td>
 <td>500</td>
 <td>0</td>
 <td>100</td>
 </tr>
 <tr>
 <td>13:51</td>
 <td>3000</td>
 <td>100</td>
 <td>0</td>
 </tr>
 <tr>
 <td>19:50</td>
 <td>3000</td>
 <td>100</td>
 <td>0</td>
 </tr>
</tbody>
</table>
 Buffer A: 10 mM Tris buffer, pH 7.4, 150 mM sodium chloride, 10 mM calcium chloride, 10 mM magnesium chloride 
 Buffer B: 0.5 M acetic acid 
 The UV detector should be programmed to read at 280 nm from 0:00 to 19:50. The y-axis should be adjusted to detect low-absorbance levels, e.g., 0 - 50 AU.</li>
<li>If an autosampler is available, program the following schedule for fraction collection. Otherwise, collect the fractions by hand as indicated when performing chromatography. 
<table border="1" cellspacing="0" cellpadding="0">
 <tbody>
 <tr>
 <td>Fraction</td>
 <td>Delay to collection from sample injection </td>
 <td>Duration of collection (min)</td>
 </tr>
 <tr>
 <td>Flow-through</td>
 <td>2.8</td>
 <td>4.1</td>
 </tr>
 <tr>
 <td>Bound</td>
 <td>9</td>
 <td>2.6</td>
 </tr>
</tbody>
</table>
</li>
</ol>
4) Prepare samples for HPLC
<ol>
<li>Prior to use in this protocol, human plasma should be depleted of the 14 most-abundant proteins using a MARS column (Agilent; Santa Clara, CA). Depleted plasma, human lactoferrin and yeast invertase should be individually trypsin-digested and desalted as previously described 1.</li>
<li>To prepare the lectin-specific lactoferrin standard, perform chromatography on 50 ug trypsin-digested lactoferrin over either the AAL or the SNA column using the method in Part 3. The bound fraction will contain lectin-specific lactoferrin glycopeptides. Neutralize the samples as in Part 4.4, and desalt as in Part 6. Resuspend the sample in 1 mL Buffer A. The resulting sample is ready for use.</li>
<li>Three experimental replicates will be performed. To prepare sample for three replicates, add 3 pmol of trypsin-digested invertase and 1 &mu;l of lectin-specific lactoferrin (either AAL-LAC or SNA-LAC) to 30 &mu;l MARS-depleted, trypsin-digested, plasma equivalents (PE). A PE is defined as the volume of non-processed/intact/original plasma from which a given amount of processed plasma (MARS-depleted and trypsin digested) was derived. Add Buffer A to the sample to reach a final volume of 330 &mu;l.</li>
<li>Add neutralization buffer (1 M Tris pH 8.0), to the sample vials that will collect eluate. The number and volume of vials you work with will depend upon the constraints of your autosampler. For the Paradigm MG4, one vial collects the flow-through and two collect the eluate. It will require approximately 1.5 mL Tris buffer to neutralize 1 mL of eluate. Target neutralized pH is 7.0-8.0; test test final pH with pH indicator paper.</li>
</ol>
5) Perform chromatography
<ol>
<li>All the blank and sample runs that follow will use the method described in Part 1. The column and buffers are at room temperature while in use. Columns are stored at 4 &deg;C, while buffers are stored at room temperature.</li>
<li>Attach either the AAL or the SNA lectin column to the HPLC system and run two blank methods (injecting only Buffer A). Ensure that the lectin column corresponds to the lectin-specific lactoferrin (Part 2).</li>
<li>Chromatograph the three plasma samples, injecting a blank in between each injected sample, for a total of 7 HPLC runs. Fractions from blank runs need not be collected.</li>
</ol>
6) Desalt collected fractions
For each fraction use one 1 mL Waters Oasis HLB SPE cartridge as follows:
<ol>
<li>Attach the number of required cartridges to vacuum manifold.</li>
<li>Wet each cartridge with 1 mL 80% ACN in 1% formic acid (vacuum gauge on manifold should read 5 - 20 lnHg).</li>
<li>Equilibrate cartridges with 1.5 mL 0.1% formic acid (vacuum gauge on manifold should read 5 - 20 lnHg)</li>
<li>Load entire volume of one sample onto one cartridge (Vacuum gauge on manifold should read 2 - 2.5 lnHg, and flow rate should not exceed 1 mL/min.)</li>
<li>Wash cartridges with 3 x 1 mL 0.1% formic acid (Vacuum gauge on manifold should read 5 - 20 lnHg)</li>
<li>Elute peptides/glycopeptides into labeled eppendorf tubes with 1 x 1 mL 80% acetonitrile in 0.1% formic acid. (Vacuum gauge on manifold should read 2 - 2.5 lnHg, and flow rate should not exceed 1 mL/min.) A single collection tube should be used for each cartridge.</li>
<li>Neutralize eluate by adding 60 &mu;l 0.5 M ammonium bicarbonate to each collection tube. Target neutralized pH is is 7.0-8.0; test final pH with pH indicator paper.</li>
<li>Concentrate eluted peptides/glycopeptides to ~50 &mu;L by running them on a vacuum centrifuge (e.g., a Thermo Savant SpeedVac) for ~2 hrs @ 35&deg;C.</li>
</ol>
7) PNGase F-digestion
<ol>
<li>Test sample pH to ensure it is between 7.0 - 8.0. If needed, add 50 mM ammonium bicarbonate to increase pH. If final sample volume is &gt; 100 &mu;l, speedvac sample until it is between 50-100 &mu;l.</li>
<li>Add 0.5 &mu;l (250 U) glycerol-free PNGase F to each sample tube and incubate at 37 &deg;C overnight.</li>
</ol>
8) Zip Tip samples
<ol>
<li>Starting with PNGase F-digested samples, ziptip each one as follows.</li>
<li>Wet ziptip with 10 &mu;l 100% acetonitrile, followed by 10 &mu;l 80% acetonitrile, 0.1% formic acid.</li>
<li>Equilibrate ziptip with 20 &mu;l 0.1% formic acid.</li>
<li>Load the sample onto the ziptip by pipetting it up and down through the ziptip matrix 10 times.</li>
<li>Wash the ziptip by pipetting 10 &mu;l 0.1% formic acid, 5 times.</li>
<li>Elute the sample into a clean eppendorf tube with 10 &mu;l 80% acetonitrile, 0.1% formic acid.</li>
<li>Repeat the elution and add the second 10 &mu;l into the same tube.</li>
<li>Speedvac the samples to a volume of &lt; 2 &mu;l.</li>
<li>Resuspend samples in 20 &mu;l 0.1% formic acid. Samples are now ready for analysis by LC-MS/MS.</li>
</ol>
9) Representative Results:
The POROS conjugation typically yields on-bead lectin concentrations in the range of 2 - 20 mg/mL. This is optimal for affinity chromatography. 
Lectin chromatography of trypsin-digested MARS-depleted human plasma typically enriches glycopeptides in the bound fraction. As the glycopeptides are PNGase F-treated prior to LC-MS/MS, they are identified as peptides with the N-linked consensus sequence, NXS/T (where X is any residue but proline), in which the asparagine has been converted to an aspartic acid through the action of PNGase F. Peptides with these characteristics are considered to be deglycosylated peptides (or "deglycopeptides"). On average the flow-through (FT) fraction from AAL or SNA chromatography contains 2-4% deglycopeptides, whereas 30-50% of the peptides recovered in the bound fraction are deglycopeptides. Typically, using a QStar Elite, 1000-1300 peptides will be identified in the FT fraction, and 200-400 peptides will be identified in the bound fraction. Two or more distinct Invertase deglycopeptides should be observed in the FT fraction and two or more distinct lactoferrin deglycopeptides in the bound fraction (Table 1).
<img src="/files/ftp_upload/1398/1398table.jpg" alt="Table 1" /> Table 1.

<ol>
	<li>Keshishian, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=multiplexed+assays+for+low+abundance+proteins+in+plasma+by+targeted+mass+spectrometry+and+stable+isotope+dilution.">multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution.</a> Mol Cell Proteomics. 6, 2212-2229 (2007).</li></ol>

This protocol provides a quick method for isolating and identifying glycopeptides in a glycan-specific manner. As glycosylation varies extensively within an organism, particularly during development and pathogenesis, this technique may be applicable to address a wide variety of questions. In particular, we are using the method to selectively enrich glycopeptides that have been modified with cancer-associated epitopes as a first step towards biomarker discovery.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Human plasma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>We make this in house</td>
</tr>
<tr>
<td>Human lactoferrin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L0520</td>
<td>Trypsin-digest before use</td>
</tr>
<tr>
<td>Yeast invertase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I0408</td>
<td>Trypsin-digest before use</td>
</tr>
<tr>
<td>Oass HLB SPE cartridges</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>WAT094225</td>
<td>1 cc volume</td>
</tr>
<tr>
<td>ZipTip pipet tips</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>ZTC1 8M0 96</td>
<td>Millipore, C18 for 10 ml pipette</td>
</tr>
<tr>
<td>Tris</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP154-1</td>
<td>99%</td>
</tr>
<tr>
<td>Ammonium bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A6141</td>
<td>99%</td>
</tr>
<tr>
<td>Calcium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C4901</td>
<td>96%</td>
</tr>
<tr>
<td>Magnesium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M8266</td>
<td>98%</td>
</tr>
<tr>
<td>Acetic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>242853</td>
<td>99.7%</td>
</tr>
<tr>
<td>pH indicator paper</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>M95903</td>
<td>Range 0-14</td>
</tr>
<tr>
<td>Acetonitrile</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>A998</td>
<td>99.9%</td>
</tr>
<tr>
<td>Formic acid</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>28905</td>
<td>99%</td>
</tr>
<tr>
<td>Phosphate-buffered saline</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14190-136</td>
<td>Without calcium and magnesium</td>
</tr>
<tr>
<td>Sodium azide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>71289</td>
<td>99.5%</td>
</tr>
<tr>
<td>PNGase F</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>P0705L</td>
<td>Glycerol-free</td>
</tr>
<tr>
<td>Vacuum-filter flasks</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SCGVU05RE</td>
<td>0.2 mm pores</td>
</tr>
<tr>
<td>POROS-AL beads</td>
<td>&nbsp;</td>
<td>Applied Biosystems</td>
<td>1-6028-02</td>
<td>&nbsp;</td>
<tr>
<td>Aleuria aurantia lectin</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>L-1390</td>
<td>&nbsp;</td>
<tr>
<td>Sambucus nigra agglutinin</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>L-1300</td>
<td>&nbsp;</td>
<tr>
<td>Sodium cyanoborohydride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>296945</td>
<td>5.0 M solution</td>
</tr>		</tbody>
		</table>
		</div>

a lectin hplc method to enrich selectively-glycosylated peptides from complex biological samples

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls&mdash;fucosylated and sialylated human lactoferrin glycopeptides&mdash;and negative controls&mdash;high mannose glycopeptides from Saccharomyces cerevisiae&mdash;that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.

Watch this Scientific Journal Video about A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples at JoVE.com

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the ...

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15.9K Views.  University of California, San Francisco - UCSF. Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.

Video: A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (&Omega;). 
Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as &Omega; is related to the overall shape and topology of the ion. The correlation between the measured drift time values and &Omega; is calculated using a calibration curve generated from calibrant proteins with defined cross-sections1.
The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions1. Several recent IM studies of both individual proteins2,3 and non-covalent protein complexes4-9, successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)10. We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical &Omega; values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects on bone repair in a unicortical defect model. Treatment of mesenchymal cells produced an increase in alkaline phosphatase activity, showed nodule formation by the cells, and increased the expression of genes for runx2, osterix, bone sialoprotein, and osteocalcin. A collagen sponge soaked with peptide promoted repair of bone defects, whereas the control was less effective. The results from this study demonstrated that mesenchymal cells treated with peptide in vitro differentiate towards osteogenesis, and, that peptides delivered in vivo using a collagen sponge promote the repair of unicortical defects.

A phage display library was used to identify peptide sequences that target bone. The objective was to investigate the effect of these peptides on mesenchymal cell differentiation and to determine their effect on bone regeneration.

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities. It is particularly useful for those compounds which possess therapeutic value but cannot be sufficiently produced or would benefit from an improved form of production. The experimental procedures involved can be subdivided into three components: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each experimental component is under continual optimization to meet the challenges and anticipate the opportunities associated with this emerging approach.
Heterologous biosynthesis begins with the identification of a genetic sequence responsible for a valuable natural product. Transferring this sequence to a heterologous host is complicated by the biosynthetic pathway complexity responsible for product formation. The antibiotic erythromycin A is a good example. Twenty genes (totaling &gt;50 kb) are required for eventual biosynthesis. In addition, three of these genes encode megasynthases, multi-domain enzymes each ~300 kDa in size. This genetic material must be designed and transferred to E. coli for reconstituted biosynthesis. The use of PCR isolation, operon construction, multi-cystronic plasmids, and electro-transformation will be described in transferring the erythromycin A genetic cluster to E. coli.
Once transferred, the E. coli cell must support eventual biosynthesis. This process is also challenging given the substantial differences between E. coli and most original hosts responsible for complex natural product formation. The cell must provide necessary substrates to support biosynthesis and coordinately express the transferred genetic cluster to produce active enzymes. In the case of erythromycin A, the E. coli cell had to be engineered to provide the two precursors (propionyl-CoA and (2S)-methylmalonyl-CoA) required for biosynthesis. In addition, gene sequence modifications, plasmid copy number, chaperonin co-expression, post-translational enzymatic modification, and process temperature were also required to allow final erythromycin A formation.
Finally, successful production must be assessed. For the erythromycin A case, we will present two methods. The first is liquid chromatography-mass spectrometry (LC-MS) to confirm and quantify production. The bioactivity of erythromycin A will also be confirmed through use of a bioassay in which the antibiotic activity is tested against Bacillus subtilis. The assessment assays establish erythromycin A biosynthesis from E. coli and set the stage for future engineering efforts to improve or diversify production and for the production of new complex natural compounds using this approach.

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The heterologous biosynthesis of erythromycin A through E. coli includes the following experimental steps: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each step will be explained in the context of the motivation, potential, and challenges in producing therapeutic natural products using E. coli as a surrogate host.

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities.  It is ...

A Method for Microinjection of Patiria minata Zygotes

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and evolution of developmental processes. The sea star, Patiria miniata, is gaining prevalence as a model system for these types of studies which were previously performed almost exclusively in the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. An advantage of these model systems is the ease of producing modified embryos in which a particular gene is up or downregulated, labeling a group of cells, or introducing a reporter gene. A single microinjection method is capable of creating a wide variety of such modified embryos. Here, we present a method for obtaining gametes from P. miniata, producing zygotes, and introducing perturbing reagents via microinjection. Healthy morphant embryos are subsequently isolated for quantitative and qualitative studies of gene function. The availability of genome and transcriptome data for this organism has increased the types of studies that are performed and the ease of executing them.

A Method for Microinjection of Patiria minata Zygotes

Methods that produce morphant embryos are essential to study developmental mechanisms and gene regulatory networks. The sea star Patiria miniata is an emerging model system for these studies. Here we present a protocol for obtaining gametes, producing cultures of embryos, and rapid microinjection of zygotes from this species.

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and ...

Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.

Using the cystic fibrosis airway as an example, the manuscript presents a comprehensive workflow comprising a combination of metagenomic and metatranscriptomic approaches to characterize the microbial and viral communities in animal-associated samples.

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect conditions over vast time periods and investigation of biological materials harbored inside could provide insight of ancient environments. To appropriately analyze these ecosystems and extract meaningful biological information from frozen soils and ice, proper collection and processing of the frozen samples is necessary. This is especially critical for microbial and DNA analyses since the communities present may be so uniquely different from modern ones. Here, a protocol is presented to successfully collect and decontaminate frozen cores. Both the absence of the colonies used to dope the outer surface and exogenous DNA suggest that we successfully decontaminated the frozen cores and that the microorganisms detected were from the material, rather than contamination from drilling or processing the cores.

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. A protocol is presented to collect and decontaminate permafrost cores of soils and ice. The absence of exogenous colonies and DNA suggest that microorganisms detected represent the material, rather than contamination from drilling or processing.

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect ...

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.

This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.