"description":"This template will help you prepare protein samples, followed by the cleanup and mass spectrometry analysis. \r\nIn bottom-up proteomics, the protein is first broken up into peptides, either by chemical or enzymatic digestion. In the first step, sample preparation is required after gel electrophoresis, liquid chromatography or affinity capture. Samples can (optionally) be enriched at the peptide level. With mass spectrometry, in the end, you can identify and characterize biological molecules.",
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><p>Depending on the different type of samples there are different mass spectrometry instrument that can be selected.</p></body></html>"
},
"position":0
},
{
"table":{
"id":622,
"created_at":"2018-11-28T07:54:48.627Z",
"updated_at":"2018-12-24T08:08:49.543Z",
"created_by_id":202,
"last_modified_by_id":202,
"name":"Commonly used mass spectrometers and their characteristics",
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><p>Differences among search engines can include the scoring algorithm, whether it considers data quality, and whether it considers all fragment ion types or only a subset. Often, the best way to select an algorithm is to examine the details regarding data-format compatibility and necessary features, test it on a data set, and compare the results to what is expected.<br><br></p></body></html>"
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><p>After the MS data is collected, the next step is to perform a database search to identify the proteins and/or peptides analyzed by MS. A number of parameters can affect the outcome of a database search, and, ultimately, search parameters should consider the history of the sample, such as organism, enzymatic or chemical digestion, and reduction/alkylation steps.</p></body></html>"
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><div style=\"text-align: justify;\">The protein is cut enzymatically into a limited number of shorter fragments during digestion and these fragments are called peptides and allow for the identification of the protein with their characteristic mass and pattern.</div></body></html>"
"text":"Dilute the gel enzyme stock solution ~1:1000 with 25 mM ammonium bicarbonate to obtain a 10 to 20 μg/mL working solution.",
"checked":false,
"checklist_id":942,
"created_at":"2018-12-20T14:15:25.671Z",
"updated_at":"2018-12-20T14:15:25.671Z",
"created_by_id":null,
"last_modified_by_id":null,
"position":0
},
{
"id":3870,
"text":"Add a sufficient amount of the gel enzyme working solution to cover gel pieces and incubate on ice for 1 hr. For a typical gel band/spot, this is ~10 to 20 μL.",
"checked":false,
"checklist_id":942,
"created_at":"2018-12-20T14:15:25.679Z",
"updated_at":"2018-12-20T14:15:25.679Z",
"created_by_id":null,
"last_modified_by_id":null,
"position":1
},
{
"id":3871,
"text":"Remove excess gel enzyme solution, then add sufficient 25 mM NH4HCO3to cover the gel pieces. This increases the pH and thereby inhibits the enzymatic digestion for those enzymes which have low pH optima.",
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body>\n<em><strong>Dithiothreitol (DTT) is a reducing agent that converts cystine’s disulfide bond into cysteine’s free sulfhydryl groups.</strong></em><br><em><strong>Iodoacetamide (IAA) is an alkylating agent that reacts with free sulfhydryl groups of cysteine residues to form S-</strong></em><strong><em>carboxyamidomethyl-cysteine, which cannot be reoxidized to form disulfide bonds. This is important for allowing trypsin maximum access to cleavage sites within the protein.</em></strong>\n</body></html>"
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><p>Here is the guideline how to extract peptides.This step can take up to several hours depending upon the volume used.</p></body></html>"
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body>\n<em><strong>Coomassiedyes(also known asCoomassie Brilliant Dyes) are a family of dyes commonly used to stain proteins in sodium dodecyl sulfate and blue native polyacrylamide gel electrophoresis (SDS-PAGE and BN-PAGE, respectively) gels.</strong></em> The gels are soaked in dye and an excess stain is then eluted with a solvent. This treatment allows the visualization of protein bands.</body></html>"
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><p>Destain in the microcentrifuge tube for <strong>30 min</strong> with <strong>100 μL</strong> of the appropriate gel destain solution with vigorous vortexing. The choice of destaining protocol depends on the stain used, and there is a specific destain recipe for each type of gel stain.The choice of protein stain depends on the protein concentration and the dynamic range of the sample. Silver and colloidal Coomassie staining (CCS) stains offer a low limit of detection (<strong>1 to 10 ng</strong>) compared to traditional Coomassie staining (<strong>0.3 to 1 μg)</strong>.</p></body></html>"
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body>\n<em><strong>Silver staining is used to detect proteins after electrophoretic separation on polyacrylamide gels.</strong></em> It combines excellent sensitivity, whilst using very simple and cheap equipment and chemicals.</body></html>"
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><p style=\"text-align: justify;\">Once you have run your gel, move it to an<strong> open UV box</strong> (be sure to wear proper UV protection - especially for your eyes!), remove it from any gel tray as plastic will block much of the UV and with a clean, sterile razor blade, slice the desired DNA fragment from the gel. Try to get as little excess gel around the band as possible. To do so, it is often important to take the excised band, lay it down on the UV box and trim the top, bottom and sides with the razor blade. This is especially important during the DNA purification step, as many kits cannot handle more than a certain total volume of gel per reaction.<br><br></p></body></html>"
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><div style=\"text-align: justify;\">The pH of the sample may be neutralized either prior to or after drying via vacuum centrifugation to remove acetonitrile and trifluoroacetic acid.<span style=\"text-align: left;\">RP-HPLC samples will likely contain aqueous trifluoroacetic acid and acetonitrile since these are the most commonly used mobile phases.</span><span style=\"text-align: left;\">The NH</span><sub style=\"text-align: left;\">4</sub><span style=\"text-align: left;\">HCO</span><sub style=\"text-align: left;\">3</sub><span style=\"text-align: left;\">solution should be prepared fresh, as the pH of the solution will increase with age at room temperature. It is possible to store for several days at <strong>4°C</strong>, but the pH should be checked prior to use.</span>\n</div></body></html>"
},
"position":0
},
{
"checklist":{
"checklist":{
"id":944,
"name":"To neutralize prior to drying:",
"step_id":4840,
"created_at":"2018-12-20T14:20:33.276Z",
"updated_at":"2018-12-20T14:20:33.276Z",
"created_by_id":null,
"last_modified_by_id":null
},
"checklist_items":[
{
"id":3878,
"text":"Add sufficient 1 M NH4HCO3, pH8.5.",
"checked":false,
"checklist_id":944,
"created_at":"2018-12-20T14:20:33.278Z",
"updated_at":"2018-12-20T14:20:33.278Z",
"created_by_id":null,
"last_modified_by_id":null,
"position":0
},
{
"id":3879,
"text":"Adjust final pH to ~8.0.",
"checked":false,
"checklist_id":944,
"created_at":"2018-12-20T14:20:33.285Z",
"updated_at":"2018-12-20T14:20:33.285Z",
"created_by_id":null,
"last_modified_by_id":null,
"position":1
},
{
"id":3880,
"text":"Then dry via vacuum centrifugation.",
"checked":false,
"checklist_id":944,
"created_at":"2018-12-20T14:20:33.292Z",
"updated_at":"2018-12-20T14:20:33.292Z",
"created_by_id":null,
"last_modified_by_id":null,
"position":2
}
]
},
"position":1
},
{
"checklist":{
"checklist":{
"id":945,
"name":"To neutralize after drying:",
"step_id":4840,
"created_at":"2018-12-20T14:21:35.471Z",
"updated_at":"2018-12-20T14:21:35.471Z",
"created_by_id":null,
"last_modified_by_id":null
},
"checklist_items":[
{
"id":3881,
"text":"Resuspend dried protein in 100 mM NH4HCO3, pH 8.5.",
"checked":false,
"checklist_id":945,
"created_at":"2018-12-20T14:21:35.474Z",
"updated_at":"2018-12-20T14:21:35.474Z",
"created_by_id":null,
"last_modified_by_id":null,
"position":0
},
{
"id":3882,
"text":"Spot an aliquot of the neutralized solution on narrow-range pH paper to validate that neutralization (to pH 8.0) has occurred.",
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body>\n<p><em><strong>Reduction of disulfide bonds can be achieved with either dithiothreitol (DTT) or tris (2-carboxyethyl)phosphine hydrochloride (TCEP).</strong></em>DTT works optimally at <strong>pH 7 to 9</strong>, TCEP is typically more efficient, works at a wider pH range (<strong>pH 2 to 11</strong>), and has no offensive odour. The concentrations and incubation times for DTT, TCEP, and IAA may vary depending on the amount of protein to be treated. Typical concentrations range from <strong>5 to 10 mM</strong> for DTT and TCEP, and <strong>10 to 50 mM</strong> for iodoacetamide. Incubation times may range from <strong>5 min to 1 hr for the reduction step</strong> and <strong>10 min to 1 hr for the alkylation step</strong>. Similarly, the reduction can be performed at room temperature, <strong>37°C or 56°C</strong>. Each parameter can be optimized depending on the nature of the protein sample.</p>\n<div><em>Optional:Following alkylation, adjustthe sample to <strong>40 mM</strong> DTT.</em></div>\n</body></html>"
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body>\n<div style=\"text-align: justify;\">Digest the protein sample into peptides using an <strong>appropriate enzyme</strong> (e.g., chymotrypsin, trypsin, LysC, or AspN) or<strong> chemical</strong> (CNBr/70% formic acid). If using trypsin, add sufficient enzyme for final trypsin:protein ratio of <strong>1:20 to 1:100</strong> (w/w). Vortex briefly, seal the tube with Parafilm and incubate with end-over-end rotation at <strong>37°C</strong> for <strong>4 to 18 hours</strong>.</div>\n<div style=\"text-align: justify;\">\n<br>The amount of trypsin needed will vary depending on the amount of protein sample present and the desired speed of digestion. To minimize trypsin autolysis, which will add extra peaks to the MS spectra, use the minimum amount of trypsin.<strong> Typically, a ratio of 1:20 (w/w) is sufficient for complete digestion of 10 μg protein within 4 hr</strong>. If protein concentration is especially high or the accessibility of trypsin to the cleavage sites is hampered (e.g., due to insolubility), it may be helpful to add another aliquot of trypsin or perform short digestion (<strong>4 hours</strong>) with LysC prior to the addition of trypsin. Finally, the activity of trypsin is enhanced in the presence of acetonitrile (<strong>10% to 50% v/v</strong>), and, thus, acetonitrile may be added to aid in the solubilization of the protein during tryptic digestion.</div>\n<div style=\"text-align: justify;\">\n<br><strong>Trypsin activity is greatest at pH 8.0</strong>. Thus, it is recommended that the pH of the solution be checked prior to the addition of the enzyme. In cases where sample volume is small, a <strong>1- to 2-μL</strong>sample may be tested using pH paper. Stop reaction by adding <strong>5 μL</strong> of<strong> 1.0%</strong> trifluoroacetic (TFA) acid (pH after TFA addition should be <strong>~2 to 3</strong>).</div>\n</body></html>"
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><p>Follow elution and collection guidelinesbelow.</p></body></html>"
"text":"If samples have been dried, then resuspended in 3 μL 1 of % SDS.Dilute IMAC–1% TFA and IMAC-FT samples each in 5 volumes of TiO2loading solution.",
"checked":false,
"checklist_id":951,
"created_at":"2018-12-20T14:51:18.467Z",
"updated_at":"2018-12-20T14:51:18.467Z",
"created_by_id":null,
"last_modified_by_id":null,
"position":0
},
{
"id":3902,
"text":"Load each of the samples onto one TiO2column (for simple samples) or three to four TiO2 columns (for complex mixtures containing ≥120 μg protein).",
"checked":false,
"checklist_id":951,
"created_at":"2018-12-20T14:51:18.476Z",
"updated_at":"2018-12-20T14:51:18.476Z",
"created_by_id":null,
"last_modified_by_id":null,
"position":1
},
{
"id":3903,
"text":"Collect TiO2flow through in microcentrifuge tubes. Do this step slowly using Combi-Syringe to apply the pressure.",
"checked":false,
"checklist_id":951,
"created_at":"2018-12-20T14:51:18.483Z",
"updated_at":"2018-12-20T14:51:18.483Z",
"created_by_id":null,
"last_modified_by_id":null,
"position":2
},
{
"id":3904,
"text":"Wash the TiO2columns using 5 μL TiO2loading solution and pool the eluates with the TIO2FT fraction from the previous step. Do this step slowly using Combi-Syringe to apply the pressure.",
"checked":false,
"checklist_id":951,
"created_at":"2018-12-20T14:51:18.491Z",
"updated_at":"2018-12-20T14:51:18.491Z",
"created_by_id":null,
"last_modified_by_id":null,
"position":3
},
{
"id":3905,
"text":"Elute the phosphopeptides using 30 μL high-pH elution solution.Use 30 μL per column and pool the eluates from the same sample. This step should be performed slowly using a Combi-Syringe to apply pressure.\r\nAcidify the eluates using 100% TFA to a pH of ~2 to 3.",
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><p>Different preparations of samples for different types of mass spectrometry.</p></body></html>"
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body>\n<div style=\"text-align: justify;\">Prepare a slurry of Oligo R3 reversed-phase resin in <strong>50%</strong> acetonitrile. Pack the Oligo R3 into a P20 narrow-bore pipet tip. Squeeze the tip of the narrow-bore P20 pipet tip to prevent the beads from leaking out. Sufficient Oligo R3 slurry should be added to make the columns <strong>~5 mm</strong> long. It is important to have a good seal on these columns, as column breakthrough will block on-line liquid chromatography systems.</div>\n<div style=\"text-align: justify;\">Load each of the phosphopeptide-containing eluates that have been enriched with the TiO<sub>2</sub>strategy onto one (for simple samples) or two to three (for complex mixtures) Oligo R3 columns depending on the amount of material.<br>Wash the Oligo R3 columns using <strong>30 μL 0.1%</strong> TFA.</div>\n</body></html>"
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body>\n<div style=\"text-align: justify;\">Prepare a TiO<sub>2</sub>bead slurry in <strong>100%</strong> acetonitrile. Pack the TiO<sub>2</sub>in StageTips that have been pre-packed with C<sub>8</sub>disks. The resulting TiO<sub>2</sub>columns should be <strong>~4 to 5 mm</strong> long.</div>\n<div style=\"text-align: justify;\"><em><strong>TiO<sub>2</sub>is light sensitive, so keep the powder in an amber or dark glass container or in a microcentrifuge tube covered with aluminium foil. When not in use, TiO<sub>2</sub>should be in a container stored in a dark place (e.g., a drawer). It should not be exposed to light for longer than necessary.</strong></em></div>\n</body></html>"
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><div>Dry each eluate containing the multiply phosphorylated peptides,monophosphopeptides, and the IMAC flow through separately using vacuum centrifugation.</div></body></html>"
"text":"Collect the column flow through in microcentrifuge tubes.",
"checked":false,
"checklist_id":950,
"created_at":"2018-12-20T14:48:32.249Z",
"updated_at":"2018-12-20T14:48:32.249Z",
"created_by_id":null,
"last_modified_by_id":null,
"position":0
},
{
"id":3898,
"text":"Wash the IMAC column with 50 μL IMAC wash solution and pool the eluate with the fraction from the previous step. This need to be done slowly using a Combi-Syringe to apply pressure.",
"checked":false,
"checklist_id":950,
"created_at":"2018-12-20T14:48:32.256Z",
"updated_at":"2018-12-20T14:48:32.256Z",
"created_by_id":null,
"last_modified_by_id":null,
"position":1
},
{
"id":3899,
"text":"Elute and collect the monophosphopeptides using 50 μl low-pH elution solution. This needs to be done quickly using a Combi-Syringe to apply pressure.",
"checked":false,
"checklist_id":950,
"created_at":"2018-12-20T14:48:32.263Z",
"updated_at":"2018-12-20T14:48:32.263Z",
"created_by_id":null,
"last_modified_by_id":null,
"position":2
},
{
"id":3900,
"text":"Elute and collect the multiply phosphorylated peptides using 70 μL high-pH elution solution. This step should be performed slowly using a Combi-Syringe to apply pressure.",
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body>\n<p>Squeeze the tip of the narrow-bore pipet tip nearly shut, as this will prevent bead loss. The Combi-Syringe is only to be used for the generation of pressure in the column, not for measuring and loading the aliquots. The Combi-Syringe is reusable and should therefore not come into contact with any solution.</p>\n<div></div>\n</body></html>"
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body>\n<p>To<span>o many IMAC beads may result in <strong>nonspecific binding of peptides</strong> because of reduced washing efficiency. A small amount of wash solution should be left in the tube with the beads, to avoid aspirating the beads.</span>\n</p>\n<div></div>\n</body></html>"
"text":"Wash IMAC (immobilized metal affinity chromatography) beads. You will need 5 to 7 μL for simple mixtures and 30 to 50 μL for complex mixtures containing ≥120 μg protein.",
"checked":false,
"checklist_id":948,
"created_at":"2018-12-20T14:44:55.376Z",
"updated_at":"2018-12-20T14:44:55.376Z",
"created_by_id":null,
"last_modified_by_id":null,
"position":0
},
{
"id":3892,
"text":"Applying 50 μL IMAC wash solution, vortexand centrifuge at low speed (e.g., 800 rpm in a benchtop microcentrifuge) at room temperature to pellet the beads.",
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><div>Down below are the guidelines for elution of proteins.<br><br>\n</div></body></html>"
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body>\n<em><strong>Disulfide (sulfur-sulfur) linkages between two cysteine residues are an integral component of the three-dimensional structure of many proteins.</strong> </em>Disulfide reducing agents are routinely used in biochemical reactions forpeptidesand proteins prior to MS analysis.</body></html>"
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><div style=\"text-align: justify;\">The protein is cut enzymatically into a limited number of shorter fragments during digestion and these fragments are called peptides and allow for the identification of the protein with their characteristic mass and pattern.</div></body></html>"
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><div style=\"text-align: justify;\">\n<em><strong>Wash affinity matrix containing the adsorbed protein sample with the wash buffer such as PBS to elute the unbound or nonspecifically bound proteins from the matrix.</strong></em>Beads may be washed by resuspending them in the wash buffer with gentle vortexing, then microcentrifuging at low speed (e.g., <strong>800 rpm</strong> in a standard microcentrifuge) at room temperature to pellet the beads, and removing the supernatant by pipetting.<br><strong>If detergent is present in the starting material, it may be necessary to increase the number of washes to remove residual detergent</strong>. Often this can be difficult and may require up to ten additional<strong> 20×</strong> volume washes (i.e., <strong>20 times</strong> the packed volume of the matrix). For most immunocapture methods, PBS is an effective wash buffer, but other neutral-pH saline buffers such as HEPES or Tris·Cl can be used. If greater stringency is required, for example, a high-salt wash or inclusion of a chaotropic agent, perform more stringent washes first followed by additional PBS washes. It is often necessary to optimize wash conditions for individual affinity-capture methods.</div></body></html>"
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><div>The processing of samples can be performed using a pipet or centrifuge, or with the aid of a vacuum manifold.</div></body></html>"
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><p>Use <strong>30 μL</strong> of RP LC-ESI-MS/MS elution solution for samples that will be analyzed. Collect the eluate in a clean tube. The samples are now ready for MS analysis.</p></body></html>"
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><div>\n<strong>Three to ten times with 200 μL 0.1% TFA</strong>. The volume required for sufficient rinsing depends on the concentration of salts and other reagents in the sample. If electrospray/nanospray is to be performed, it is advisable to subsequently rinse the column three times with <strong>200 μL</strong> water to remove the TFA, which may interfere with ionization in electrospray/nanospray.</div></body></html>"
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><p>Follow guidelinesfor sample preparation.</p></body></html>"
