This work was supported by German Research Foundation STE 1765/3-1 (A.S.) and German Ministry for Education and Research 03IPT614A (C.G.). We thank Reinhard Lommel and Petra Bu&#223;e for their excellent technical support as well as Karin Johansson and Christina Hentzschel for help with organization and execution of anthropometric measurements

The protocol was approved by the local ethics committee for human research (protocol number EA1/114/10).
1. Blood Processing
<ol>
	<li>Collect venous blood between 07:00 and 08:00 am after an overnight fast from a forearm vein and process according to standard procedure or the RAPID method. Instruct the subjects to not exercise or smoke before blood withdrawal.</li>
	<li>For standard processing, collect blood in chilled EDTA-containing tubes and centrifuge within 10 min at 3,000 x g for 10 min at 4 &#176;C. Collect the supernatant and keep at -80 &#176;C until further processing by radioimmunoassay.</li>
	<li>For RAPID processing, immediately dilute blood (within 1 min after blood withdrawal) 1:10 in ice-cold buffer (pH 3.6) containing 0.1 M ammonium acetate, 0.5 M NaCl and enzyme inhibitors (diprotin A, E-64-d, antipain, leupeptin, chymostatin, 1 &#181;g/ml). Then, centrifuge within 10 min at 3,000 x g for 10 min at 4 &#176;C and collect supernatant using a pipette in polypropylene tubes as detailed before in rats6.
	<ol>
		<li>Charge chromatography cartridges (360 mg, 55-105 &#181;m) with 100% acetonitrile (rate 10 ml/min), equilibrate with 0.1% trifluoroacetate (TFA, rate 10 ml/min) and load with the supernatant at a constant rate of 1 ml/min using a syringe pump.</li>
		<li>Thereafter, wash cartridges with 3 ml 0.1% TFA (rate 10 ml/min) and slowly elute with 2 ml 70% acetonitrile containing 0.1% TFA (2 ml/min).</li>
		<li>Dry eluted samples using vacuum centrifugation and store at -80 &#176;C until further processing by radioimmunoassay. 
		NOTE: Conduct all steps in polypropylene (RAPID processing) and borosilicate (radioimmunoassay) tubes that exhibit significantly lower surface binding properties and thus minimize peptide loss for most of the peptides studied before26.</li>
	</ol>
	</li>
</ol>
2. Measurements
NOTE: Steps in this section should be performed in a laboratory certified for work with radioactive material. Standard precautions for the work with 125I should be taken.
<ol>
	<li>Recovery of Radiolabeled Peptides
	<ol>
		<li>Obtain 125I-radiolabeled human peptides (e.g. acyl-ghrelin, GLP-1, glucagon, insulin, kisspeptin, leptin, nesfatin-1, PYY3-36 and somatostatin-28).</li>
		<li>Keep peptides in powder form until the experiment, then freshly dilute in 0.1% acetic acid (~100,000 cpm per ml).</li>
		<li>For standard blood processing, directly after blood withdrawal into chilled EDTA containing tubes, transfer 1 ml of blood into a tube with 50 &#181;l of radiolabel containing 3,000-6,000 cpm (counted directly before the experiment starts).</li>
		<li>For RAPID processing, transfer 1 ml blood of EDTA containing blood into a tube containing 9 ml RAPID buffer (for composition see 1.3) and 500 &#181;l radiolabel containing 30,000-60,000 cpm. Due to the 1:10 dilution use 10-times higher volume of radiolabel for RAPID processing.</li>
		<li>Afterwards, process samples as described in steps 1.2 to 1.3.2.</li>
		<li>For the recovery experiments, do not dry samples by vacuum centrifugation and do not store at -80 &#176;C. Instead, assess recovery of radioactively labeled peptides directly afterwards using a gamma counter.</li>
		<li>Measure the whole supernatant in standard samples, while in RAPID samples analyze 1/10 of the total volume to obtain comparability of the amount of radiolabel used. For measurement, transfer the supernatant in tubes fitting into the gamma counter and assess the counts per minute</li>
		<li>As a 100% standard, use two samples with 50 &#181;l of 125I-radiolabeled peptide that do not undergo processing. Measure at the same time with other samples as described in 2.1.7.</li>
		<li>Perform the experiment five to six times for each peptide.</li>
	</ol>
	</li>
	<li>High Performance Liquid Chromatography of Radiolabeled Ghrelin
	<ol>
		<li>Withdraw blood in chilled EDTA containing tubes and transfer 1 ml to tubes containing 200 &#181;l radiolabeled-acyl ghrelin containing 15,000-20,000 cpm (counted directly before the experiment starts).</li>
		<li>For RAPID processing, transfer 1 ml blood into a tube containing 9 ml RAPID buffer (for composition see 1.3) and 200 &#181;l radiolabeled acyl ghrelin containing 15,000-20,000 cpm.</li>
		<li>Afterwards, process samples as described in steps 1.2 to 1.3.2.</li>
		<li>For further analysis by reverse phase HPLC, directly load samples onto a stable bond C18 column (2.1 mm x 50 mm, 1.8 &#181;m) equilibrated in 17% acetonitrile in water (both supplemented with 0.1% TFA).</li>
		<li>After 5 min equilibration, use a gradient from 17-40% acetonitrile to elute the sample in 40 min (flow rate of 1 ml/min).</li>
		<li>Collect fractions of 1 ml every minute and analyze radioactivity using a gamma counter.</li>
		<li>In a separate experiment, load 200 &#181;l radiolabeled acyl ghrelin containing 15,000-20,000 cpm onto the column directly and perform HPLC as described in steps 2.2.4 to 2.2.6.</li>
	</ol>
	</li>
	<li>Radioimmunoassay
	<ol>
		<li>For radioimmunoassay, thaw frozen supernatants (standard processing) and vacuum dried powder (RAPID method) at room temperature.</li>
		<li>Immediately before radioimmunoassay, re-suspend dry RAPID samples in double distilled H2O according to the original volume of plasma (500 &#181;l).</li>
		<li>Assess kisspeptin and total (including both desacyl and acyl ghrelin) as well as acyl ghrelin as described before12,27 using commercial radioimmunoassays according to the manufacturers&#39; protocols. Use borosilicate tubes that allow stable pellet formation.
		<ol>
			<li>On day one, incubate the samples with assay buffer and primary antibody (e.g. anti-ghrelin) in the dilution provided by the manufacturer for a period of 24 hr.</li>
			<li>On day two, add the 125I tracer (e.g. 125I-ghrelin), vortex and incubate for a period of 24 hr.</li>
			<li>On day three, add the precipitating reagent, vortex and incubate as recommended by the manufacturer. Then, centrifuge tubes at 3,000 x g for 20 min at 4 &#176;C. Remove the supernatants and count radioactivity in the pellets using a gamma counter</li>
		</ol>
		</li>
		<li>Calculate desacyl ghrelin as the difference of total minus acyl ghrelin. Assess the acyl/desacyl ghrelin ratio by dividing acyl by desacyl ghrelin for each individual sample.</li>
		<li>Process all samples &#8211; if possible&#160;&#8211; in one batch to avoid inter-assay variability. The intra-assay variability in the present experiment was &#60;8% for kisspeptin, &#60;7% for total and &#60;9% for acyl ghrelin.</li>
	</ol>
	</li>
</ol>
3. Statistical Analysis
<ol>
	<li>Determine the distribution of the data using the Kolmogorov-Smirnov test. Express data as mean &#177; standard error of mean (SEM).</li>
	<li>Assess differences between two groups by t-test. Assess differences between multiple groups by all pair-wise multiple comparison procedures (Tukey post hoc test) or two-way ANOVA followed by Holm-Sidak method.</li>
	<li>Consider p&#160;&#60;0.05 significant and perform analyses using a statistics program.</li>
</ol>

<ol>
	<li>Finucane, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=National,+regional,+and+global+trends+in+body-mass+index+since+1980:+systematic+analysis+of+health+examination+surveys+and+epidemiological+studies+with+960+country-years+and+9.1+million+participants.">National, regional, and global trends in body-mass index since 1980: systematic analysis of health examination surveys and epidemiological studies with 960 country-years and 9.1 million participants.</a> Lancet. 377 (9765), 557-567 (2011).</li><li>James, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidemiology+of+obesity:+the+size+of+the+problem.">The epidemiology of obesity: the size of the problem.</a> J Intern Med. 263 (4), 336-352 (2008).</li><li>Stengel, A., Tach&#233;, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastric+peptides+and+their+regulation+of+hunger+and+satiety.">Gastric peptides and their regulation of hunger and satiety.</a> Curr Gastroenterol Rep. 14 (6), 480-488 (2012).</li><li>Hussain, S. S., Bloom, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+of+food+intake+by+the+gut-brain+axis:+implications+for+obesity.">The regulation of food intake by the gut-brain axis: implications for obesity.</a> Int J Obes (Lond). 37 (5), 625-633 (2013).</li><li>Hosoda, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimum+collection+and+storage+conditions+for+ghrelin+measurements:+octanoyl+modification+of+ghrelin+is+rapidly+hydrolyzed+to+desacyl+ghrelin+in+blood+samples).">Optimum collection and storage conditions for ghrelin measurements: octanoyl modification of ghrelin is rapidly hydrolyzed to desacyl ghrelin in blood samples).</a> Clin Chem. 50 (6), 1077-1080 (2004).</li><li>Stengel, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RAPID+method+for+blood+processing+yields+new+insight+in+plasma+concentrations+and+molecular+forms+of+circulating+gut+peptides.">The RAPID method for blood processing yields new insight in plasma concentrations and molecular forms of circulating gut peptides.</a> Endocrinology. 150 (11), 5113-5118 (2009).</li><li>Stengel, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide+differentially+decreases+plasma+acyl+and+desacyl+ghrelin+levels+in+rats:+Potential+role+of+the+circulating+ghrelin-acylating+enzyme+GOAT.">Lipopolysaccharide differentially decreases plasma acyl and desacyl ghrelin levels in rats: Potential role of the circulating ghrelin-acylating enzyme GOAT.</a> Peptides. 31 (9), 1689-1696 (2010).</li><li>Stengel, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold+ambient+temperature+reverses+abdominal+surgery-induced+delayed+gastric+emptying+and+decreased+plasma+ghrelin+levels+in+rats.">Cold ambient temperature reverses abdominal surgery-induced delayed gastric emptying and decreased plasma ghrelin levels in rats.</a> Peptides. 31, 2229-2235 (2010).</li><li>Stengel, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+administration+of+pan-somatostatin+agonist+ODT8-SST+prevents+abdominal+surgery-induced+inhibition+of+circulating+ghrelin,+food+intake+and+gastric+emptying+in+rats.">Central administration of pan-somatostatin agonist ODT8-SST prevents abdominal surgery-induced inhibition of circulating ghrelin, food intake and gastric emptying in rats.</a> Neurogastroenterol Motil. 23 (7), e294-e308 (2011).</li><li>Stengel, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abdominal+surgery+inhibits+circulating+acyl+ghrelin+and+ghrelin-O-acyltransferase+levels+in+rats:+role+of+the+somatostatin+receptor+subtype+2.">Abdominal surgery inhibits circulating acyl ghrelin and ghrelin-O-acyltransferase levels in rats: role of the somatostatin receptor subtype 2.</a> Am J Physiol Gastrointest Liver Physiol. 301, G239-G248 (2011).</li><li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+injection+of+urocortin+1+induces+a+CRF2+mediated+increase+in+circulating+ghrelin+and+glucose+levels+through+distinct+mechanisms+in+rats.">Intravenous injection of urocortin 1 induces a CRF2 mediated increase in circulating ghrelin and glucose levels through distinct mechanisms in rats.</a> Peptides. 39, 164-170 (2013).</li><li>Goebel-Stengel, M., Stengel, A., Wang, L., Tach&#233;, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orexigenic+response+to+tail+pinch:+role+of+brain+NPY(1)+and+corticotropin+releasing+factor+receptors.">Orexigenic response to tail pinch: role of brain NPY(1) and corticotropin releasing factor receptors.</a> Am J Physiol Regul Integr Comp Physiol. 306 (3), R164-R174 (2014).</li><li>Goebel, M., Stengel, A., Wang, L., Reeve, J., Tach&#233;, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide+increases+plasma+levels+of+corticotropin-releasing+hormone+in+rats.">Lipopolysaccharide increases plasma levels of corticotropin-releasing hormone in rats.</a> Neuroendocrinology. 93 (3), 165-173 (2011).</li><li>Banfi, G., Salvagno, G. L., Lippi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+ethylenediamine+tetraacetic+acid+(EDTA)+as+in+vitro+anticoagulant+for+diagnostic+purposes.">The role of ethylenediamine tetraacetic acid (EDTA) as in vitro anticoagulant for diagnostic purposes.</a> Clin Chem Lab Med. 45 (5), 565-576 (2007).</li><li>Stengel, A., Wang, L., Goebel-Stengel, M., Tach&#233;, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrally+injected+kisspeptin+reduces+food+intake+by+increasing+meal+intervals+in+mice.">Centrally injected kisspeptin reduces food intake by increasing meal intervals in mice.</a> Neuroreport. 22 (5), 253-257 (2011).</li><li>De Bond, J. A., Smith, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kisspeptin+and+energy+balance+in+reproduction.">Kisspeptin and energy balance in reproduction.</a> Reproduction. 147 (3), R53-R63 (2014).</li><li>Wren, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ghrelin+enhances+appetite+and+increases+food+intake+in+humans.">Ghrelin enhances appetite and increases food intake in humans.</a> J Clin Endocrinol Metab. 86 (12), 5992 (2001).</li><li>Schulman, J. L., Carleton, J. L., Whitney, G., Whitehorn, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+glucagon+on+food+intake+and+body+weight+in+man.">Effect of glucagon on food intake and body weight in man.</a> J Appl Physiol. 11 (3), 419-421 (1957).</li><li>Steinert, R. E., Poller, B., Castelli, M. C., Drewe, J., Beglinger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+administration+of+glucagon-like+peptide+1+or+peptide+YY+3-36+affects+food+intake+in+healthy+male+subjects.">Oral administration of glucagon-like peptide 1 or peptide YY 3-36 affects food intake in healthy male subjects.</a> Am J Clin Nutr. 92 (4), 810-817 (2010).</li><li>Dewan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+insulin-induced+hypoglycaemia+on+energy+intake+and+food+choice+at+a+subsequent+test+meal.">Effects of insulin-induced hypoglycaemia on energy intake and food choice at a subsequent test meal.</a> Diabetes Metab Res Rev. 20 (5), 405-410 (2004).</li><li>Schlogl, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+24-hour+ad+libitum+food+intake+is+associated+with+lower+plasma+irisin+concentrations+the+following+morning+in+adult+humans.">Increased 24-hour ad libitum food intake is associated with lower plasma irisin concentrations the following morning in adult humans.</a> Appetite. 90, 154-159 (2015).</li><li>Heymsfield, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+leptin+for+weight+loss+in+obese+and+lean+adults:+a+randomized,+controlled,+dose-escalation+trial.">Recombinant leptin for weight loss in obese and lean adults: a randomized, controlled, dose-escalation trial.</a> JAMA. 282 (16), 1568-1575 (1999).</li><li>Shimizu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+administration+of+nesfatin-1+reduces+food+intake+in+mice:+the+leptin-independent+mechanism.">Peripheral administration of nesfatin-1 reduces food intake in mice: the leptin-independent mechanism.</a> Endocrinology. 150, 662-671 (2009).</li><li>Batterham, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+food+intake+in+obese+subjects+by+peptide+YY3-36.">Inhibition of food intake in obese subjects by peptide YY3-36.</a> N Engl J Med. 349 (10), 941-948 (2003).</li><li>Shibasaki, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antagonistic+effect+of+somatostatin+on+corticotropin-releasing+factor-induced+anorexia+in+the+rat.">Antagonistic effect of somatostatin on corticotropin-releasing factor-induced anorexia in the rat.</a> Life Sci. 42 (3), 329-334 (1988).</li><li>Goebel-Stengel, M., Stengel, A., Tach&#233;, Y., Reeve, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+using+the+optimal+plasticware+and+glassware+in+studies+involving+peptides.">The importance of using the optimal plasticware and glassware in studies involving peptides.</a> Anal Biochem. 414 (1), 38-46 (2011).</li><li>Smets, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+plasma+levels+of+metastin+in+early+pregnancy+are+associated+with+small+for+gestational+age+neonates.">Decreased plasma levels of metastin in early pregnancy are associated with small for gestational age neonates.</a> Prenat Diagn. 28 (4), 299-303 (2008).</li><li>Kojima, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ghrelin+is+a+growth-hormone-releasing+acylated+peptide+from+stomach.">Ghrelin is a growth-hormone-releasing acylated peptide from stomach.</a> Nature. 402 (6762), 656-660 (1999).</li><li>Stengel, A., Yin Tach&#233;, Y., Yang, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=the+Gastric+X/A-like+Cell+as+Possible+Dual+Regulator+of+Food+Intake.">the Gastric X/A-like Cell as Possible Dual Regulator of Food Intake.</a> J Neurogastroenterol Motil. 18 (2), 138-149 (2012).</li><li>Inhoff, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Desacyl+ghrelin+inhibits+the+orexigenic+effect+of+peripherally+injected+ghrelin+in+rats.">Desacyl ghrelin inhibits the orexigenic effect of peripherally injected ghrelin in rats.</a> Peptides. 29, 2159-2168 (2008).</li><li>Hirayama, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrasting+effects+of+ghrelin+and+des-acyl+ghrelin+on+the+lumbo-sacral+defecation+center+and+regulation+of+colorectal+motility+in+rats.">Contrasting effects of ghrelin and des-acyl ghrelin on the lumbo-sacral defecation center and regulation of colorectal motility in rats.</a> Neurogastroenterol Motil. 22 (10), 1124-1131 (2011).</li><li>Horikoshi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dramatic+elevation+of+plasma+metastin+concentrations+in+human+pregnancy:+metastin+as+a+novel+placenta-derived+hormone+in+humans.">Dramatic elevation of plasma metastin concentrations in human pregnancy: metastin as a novel placenta-derived hormone in humans.</a> J Clin Endocrinol Metab. 88 (2), 914-919 (2003).</li><li>Yang, Y. U., Xiong, X. Y., Yang, L. I., Xie, L., Huang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+of+kisspeptin+levels+in+girls+with+idiopathic+central+precocious+puberty+and+its+significance.">Testing of kisspeptin levels in girls with idiopathic central precocious puberty and its significance.</a> Exp Ther Med. 9 (6), 2369-2373 (2015).</li><li>Hosoda, H., Kojima, M., Matsuo, H., Kangawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ghrelin+and+des-acyl+ghrelin:+two+major+forms+of+rat+ghrelin+peptide+in+gastrointestinal+tissue.">Ghrelin and des-acyl ghrelin: two major forms of rat ghrelin peptide in gastrointestinal tissue.</a> Biochem Biophys Res Commun. 279 (3), 909-913 (2000).</li><li>Raff, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+and+active+ghrelin+in+developing+rats+during+hypoxia.">Total and active ghrelin in developing rats during hypoxia.</a> Endocrine. 21 (2), 159-161 (2003).</li><li>Evans, M. J., Livesey, J. H., Ellis, M. J., Yandle, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+anticoagulants+and+storage+temperatures+on+stability+of+plasma+and+serum+hormones.">Effect of anticoagulants and storage temperatures on stability of plasma and serum hormones.</a> Clin Biochem. 34 (2), 107-112 (2001).</li><li>Nabuchi, Y., Fujiwara, E., Kuboniwa, H., Asoh, Y., Ushio, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stability+and+degradation+pathway+of+recombinant+human+parathyroid+hormone:+deamidation+of+asparaginyl+residue+and+peptide+bond+cleavage+at+aspartyl+and+asparaginyl+residues.">The stability and degradation pathway of recombinant human parathyroid hormone: deamidation of asparaginyl residue and peptide bond cleavage at aspartyl and asparaginyl residues.</a> Pharm Res. 14 (12), 1685-1690 (1997).</li><li>White, A., Handler, P., Smith, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Biochemistry. , (1973).</li></ol>

The authors have nothing to disclose.

We reported before that the RAPID method for blood processing improved the recovery for 11/12 peptides compared to standard blood processing in rats6. In the present study we have shown that this method is also suited for the use in humans. Following RAPID processing, the recovery for 9 of 9 125I-labeled peptides tested was improved compared to standard blood processing (EDTA blood on ice). The observed improvement ranged from 19-39% which is likely to be relevant, especially under conditions when o...

In light of the worldwide increasing prevalence of obesity1,2, research in the field of food intake regulation is gaining importance. While so far only one peptide is known that is peripherally produced and centrally acting to stimulate food intake, namely ghrelin3, within the past decades, a broad range of peptides has been identified that reduce food intake, e.g. leptin, peptide YY (PYY) and also glucagon-like peptide-1 (GLP-1) and insulin4, Therefore, in studies investigating the regulatory mechanisms of hunger and satiety peptide levels are often assessed and at the same time, it is assumed that the peptide studied is stable and recovered at high yields during plasma formation. However, very often this is not the case due to rapid endogenous breakdown as shown before for e.g. ghrelin which is degraded from acyl to desacyl ghrelin5. Therefore, we recently described the RAPID method for blood processing in rats employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution6. This method improved the recovery for 11 of 12 peptides tested and allowed for the determination of the correct circulating molecular form compared to standard blood processing (EDTA blood on ice)6. This method has been used in several subsequent studies7-12 for the detection of circulating ghrelin as well as corticotropin-releasing factor13. Therefore, the method has proven useful for peptide research in rodents. However, since rodent studies are not always translatable to another species, the method should be established for the use in human blood as well.
The aim of the present study was to test the RAPID method for blood processing in humans compared to standard blood processing, EDTA blood on ice, which is widely recommended14 and frequently used in the clinical as well as research setting. We tested the recovery of a selection of 125I-labeled peptides involved in the regulation of food intake including established peptides as well as new candidates recently suggested to play a role in feeding regulation (effects on food intake are shown in Table 1) following processing with both methods. Hormones were also chosen to represent peptides of different length and charge (Table 2). Moreover, for ghrelin we investigated the molecular form(s) following the standard and RAPID method. Lastly, we assessed endogenous ghrelin (acyl and desacyl ghrelin) as well as kisspeptin levels, a peptide also recently suggested to play a role in the regulation of food intake15,16 following RAPID or standard processing. In addition, we also investigated these peptide levels in a population of subjects with a wide range of body mass index (ranging from 10.2-67.6 kg/m2) to study possible differences related to chronically altered body weight.
<img alt="Table 1" src="/files/ftp_upload/53959/53959table1.jpg" />
<img alt="Table 2" src="/files/ftp_upload/53959/53959table2.jpg" />
Diagnosis, Assessment, and Plan: 
Study participants 
All study participants were newly hospitalized patients (inclusion was within two days of admission to the hospital) of the Division of Psychosomatic Medicine at Charit&#233;-Universit&#228;tsmedizin Berlin and gave written informed consent. To avoid any impact of gender only female patients were included. A total of 42 subjects participated in this study and were divided into three groups: normal weight (BMI 18.5-25 kg/m2, n = 12), anorexia nervosa (BMI &#60;17.5 kg/m2, n = 15) and obesity (BMI &#62;30 kg/m2, n = 15). Anorexic and obese patients were diagnosed according to the International Classification of Diseases-10 and hospitalized for weight gain (anorexia nervosa) or weight reduction (obesity), respectively. All normal weight patients were hospitalized exclusively due to somatoform symptoms without relevant somatic disorders. Patients with gastrointestinal somatoform symptoms or a history of gastrointestinal surgery were excluded. Exclusion criteria also encompassed an age &#60;18 years, current pregnancy and untreated psychotic diseases. Blood collection was performed on day 2 or 3 after hospital admission before receiving dietary treatment in order to increase or reduce body weight, respectively. Anthropometric parameters were assessed on the same day.

<table><tbody><tr><td>Diprotin A</td><td>Peptides International, Louisville, KY, USA</td><td>IDP-4132</td><td></td></tr><tr><td>E-64-d</td><td>Peptides International, Louisville, KY, USA</td><td>IED-4321-v</td><td></td></tr><tr><td>Antipain</td><td>Peptides International, Louisville, KY, USA</td><td>IAP-4062</td><td></td></tr><tr><td>Leupeptin</td><td>Peptides International, Louisville, KY, USA</td><td>ILP-4041</td><td></td></tr><tr><td>Chymostatin</td><td>Peptides International, Louisville, KY, USA</td><td>ICY-4063</td><td></td></tr><tr><td>Sep-Pak C18 cartridges</td><td>Waters Corporation, Milford, MA, USA</td><td>WAT051910</td><td>360 mg, 55-105 &amp;micro;m</td></tr><tr><td>Acyl-ghrelin</td><td>Millipore, Billerica, MA, USA</td><td>9088-HK</td><td>Radioactive</td></tr><tr><td>GLP-1</td><td>Millipore, Billerica, MA, USA</td><td>9035-HK</td><td>Radioactive</td></tr><tr><td>Glucagon</td><td>Millipore, Billerica, MA, USA</td><td>9030</td><td>Radioactive</td></tr><tr><td>Insulin</td><td>Millipore, Billerica, MA, USA</td><td>9011S</td><td>Radioactive</td></tr><tr><td>Leptin</td><td>Millipore, Billerica, MA, USA</td><td>9081-HK</td><td>Radioactive</td></tr><tr><td>Kisspeptin-10</td><td>Phoenix Pharmaceuticals, Burlingame, CA, USA</td><td>T-048-56</td><td>Radioactive</td></tr><tr><td>Nesfatin-1</td><td>Phoenix Pharmaceuticals, Burlingame, CA, USA</td><td>T-003-26</td><td>Radioactive</td></tr><tr><td>PYY&lt;sub&gt;3-36&lt;/sub&gt;</td><td>Phoenix Pharmaceuticals, Burlingame, CA, USA</td><td>T-059-02&amp;nbsp;</td><td>Radioactive</td></tr><tr><td>Somatostatin-28</td><td>Phoenix Pharmaceuticals, Burlingame, CA, USA</td><td>T-060-16&amp;nbsp;</td><td>Radioactive</td></tr><tr><td>ZORBAX Rapid Resolution HT SB-C18 column</td><td>Agilent Technologies, Santa Clara, CA, USA</td><td>822700-902</td><td>2.1 mm x 50 mm, 1.8 &amp;micro;m</td></tr><tr><td>Agilent 1200 LC&amp;nbsp;</td><td>Agilent Technologies, Santa Clara, CA, USA</td><td></td><td>HPLC, several components, therefore no single catalog number</td></tr><tr><td>Kisspeptin&amp;nbsp; RIA</td><td>Phoenix Pharmaceuticals, Burlingame, CA, USA</td><td># RK-048-56</td><td>Radioactive</td></tr><tr><td>Total ghrelin RIA</td><td>Millipore, Billerica, MA, USA</td><td># GHRT-89HK&amp;nbsp;</td><td>Radioactive</td></tr><tr><td>Active ghrelin RIA</td><td>Millipore, Billerica, MA, USA</td><td># GHRA-88HK</td><td>Radioactive</td></tr><tr><td>SigmaStat 3.1</td><td>Systat Software, San Jose, CA, USA</td><td>online download</td><td></td></tr></tbody></table>

a rapid method for blood processing to increase the yield of plasma peptide levels in human blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

RAPID blood processing increases the yield of 125I-radiolabeled peptides in human blood compared to standard blood processing. 
After standard blood processing (EDTA blood on ice), the recovery of radiolabeled peptides was ~60% in 9/9 peptides (ranging from 48-68%, Figure 1A-K). RAPID processing improved the yield in all 125I-labeled peptides, namely in somatostatin-28 (+39%, Figure 1A), glucagon-like peptide-1 ...

Watch this Scientific Journal Video about A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood at JoVE.com

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

a-rapid-method-for-blood-processing-to-increase-yield-plasma-peptide

Research

JoVE Journal

Immunology and Infection

8.8K Views.  Charité Universitätsmedizin Berlin, Campus Benjamin Franklin.  RAPID bloed verwerkingsmethode kan worden gebruikt bij mensen en hogere opbrengsten peptide niveaus en maakt de beoordeling van de juiste moleculaire vorm. Daarom zal deze methode een waardevol instrument in peptide onderzoek. 

Video: A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

Genetics

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

Metallic implants, especially titanium implants, are widely used in clinical applications. Tissue in-growth and integration to these implants in the tissues are important parameters for successful clinical outcomes. In order to improve tissue integration, porous metallic implants have being developed. Open porosity of metallic foams is very advantageous, since the pore areas can be functionalized without compromising the mechanical properties of the whole structure. Here we describe such modifications using porous titanium implants based on titanium microbeads. By using inherent physical properties such as hydrophobicity of titanium, it is possible to obtain hydrophobic pore gradients within microbead based metallic implants and at the same time to have a basement membrane mimic based on hydrophilic, natural polymers. 3D pore gradients are formed by synthetic polymers such as Poly-L-lactic acid (PLLA) by freeze-extraction method. 2D nanofibrillar surfaces are formed by using collagen/alginate followed by a crosslinking step with a natural crosslinker (genipin). This nanofibrillar film was built up by layer by layer (LbL) deposition method of the two oppositely charged molecules, collagen and alginate. Finally, an implant where different areas can accommodate different cell types, as this is necessary for many multicellular tissues, can be obtained. By, this way cellular movement in different directions by different cell types can be controlled. Such a system is described for the specific case of trachea regeneration, but it can be modified for other target organs. Analysis of cell migration and the possible methods for creating different pore gradients are elaborated. The next step in the analysis of such implants is their characterization after implantation. However, histological analysis of metallic implants is a long and cumbersome process, thus for monitoring host reaction to metallic implants in vivo an alternative method based on monitoring CGA and different blood proteins is also described. These methods can be used for developing in vitro custom-made migration and colonization tests and also be used for analysis of functionalized metallic implants in vivo without histology.

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

In this video, we will demonstrate modification techniques for porous metallic implants to improve their functionality and to control cell migration. Techniques include development of pore gradients to control cell movement in 3D and production of basement membrane mimics to control cell movement in 2-D. Also, a HPLC-based method for monitoring implant integration in-vivo via analysis of blood proteins is described.

Metallic implants, especially titanium implants, are widely used in  clinical applications. Tissue in-growth and integration to these implants in  the ...

Bioengineering

Peptide and Protein Quantification Using Automated Immuno-MALDI (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a result of the direct detection of the mass-to-charge ratio of each target molecule. However, MS-based proteomics, like most other analytical techniques, has a bias towards measuring high-abundance analytes, so it is challenging to achieve detection limits of low ng/mL or pg/mL in complex samples, and this is the concentration range for many disease-relevant proteins in biofluids such as human plasma. To assist in the detection of low-abundance analytes, immuno-enrichment has been integrated into the assay to concentrate and purify the analyte before MS measurement, significantly improving assay sensitivity. In this work, the immuno- Matrix-Assisted Laser Desorption/Ionization (iMALDI) technology is presented for the quantification of proteins and peptides in biofluids, based on immuno-enrichment on beads, followed by MALDI-MS measurement without prior elution. The anti-peptide antibodies are functionalized on magnetic beads, and incubated with samples. After washing, the beads are directly transferred onto a MALDI target plate, and the signals are measured by a MALDI-Time of Flight (MALDI-TOF) instrument after the matrix solution has been applied to the beads. The sample preparation procedure is simplified compared to other immuno-MS assays, and the MALDI measurement is fast. The whole sample preparation is automated with a liquid handling system, with improved assay reproducibility and higher throughput. In this article, the iMALDI assay is used for determining the peptide angiotensin I (Ang I) concentration in plasma, which is used clinically as readout of plasma renin activity for the screening of primary aldosteronism (PA).

Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

A protocol for the protein quantification in complex biological fluids using automated immuno-MALDI (iMALDI) technology is presented.

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a ...

Chemistry

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Biology

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

Production of Autologous Platelet-Rich Plasma for Boosting In Vitro Human Fibroblast Expansion

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is required. However, cell culture using xenogenic or allogenic culture media has some disadvantages (i.e., risk of infectious agent transmission or slow cell expansion). Here, an autologous culture system is developed for the expansion of human skin fibroblast cells in vitro using a patient&#8217;s own platelet-rich plasma (PRP). Human dermal fibroblasts are isolated from the patient while undergoing abdominoplasty. Cultures are followed for up to 7 days using a medium supplemented with either fetal bovine serum (FBS) or PRP. Blood cell content in PRP preparations, proliferation, and fibroblast differentiation are assessed. This protocol describes the method for obtaining a standardized, non-activated preparation of PRP using a dedicated medical device. The preparation requires only a medical device (CuteCell-PRP) and centrifuge. This device is suitable under sufficient medical practice conditions and is a one-step, apyrogenic, and sterile closed system that requires a single, soft spin centrifugation of 1,500 x g for 5 min. After centrifugation, the blood components are separated, and the platelet-rich plasma is easily collected. This device allows a quick, consistent, and standardized preparation of PRP that can be used as a cell culture supplement for in vitro expansion of human cells. The PRP obtained here contains a 1.5-fold platelet concentration compared to whole blood together, with a preferential removal of red and white blood cells. It is shown that PRP presents a boosting effect in cell proliferation compared to FBS (7.7x) and that fibroblasts are activated upon PRP treatment.

This protocol presents a device that produces PRP to boost the in vitro expansion of cells in a 100% autologous fibroblast culture system.

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is ...

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

Screening Peptides that Activate MRGPRX2 using Engineered HEK Cells

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new therapeutics. Mas related G-protein receptor-X2 (MRGPRX2) is an important receptor that regulates mast cell activation and, thus, directs the general immune response. Numerous ligands for MRGPRX2 have been identified and include endogenous peptides like PAMPs, defensins, LL-37 and other protein fragments (i.e., degraded albumin). Further identification of MRGPRX2 specific ligands requires the screening of a large number of peptides (i.e., peptide library); however, mast cells are difficult and expensive to maintain in vitro and, therefore, not economical to use for screening large numbers of molecules. The present paper demonstrates a method to design, develop, and screen a library of small peptide molecules using MRGPRX2 expressing HEK cells. This cell line is relatively easy and inexpensive to maintain and can be used for in vitro high-throughput analysis. A calcium sensitive Fura-2 fluorescent dye to mark intracellular calcium flux upon activation was used to monitor the activation. The ratio of fluorescence intensity of Fura-2 at 510 nm against excitation wavelengths of 340 and 380 nm was used to calculate calcium concentration. The peptide library used to verify this system was based on the endogenous proadrenomedullin N-terminal 12 (PAMP-12) secretagogue, which is known to bind MRGPRX2 with high specificity and affinity. Subsequent peptides were generated through amino acid truncation and alanine scanning techniques applied to PAMP-12. The method described here is simple and inexpensive yet robust for screening a large library of compounds to identify binding domains and other important parameters that play an important role in receptor activation.

Techniques for generating a library of short peptides that can activate mast cells via the MRGPRX2 receptor are described. Associated techniques are easy, inexpensive, and can be extended to other cell receptors.

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

Enrichment of Astrocyte-Derived Extracellular Vesicles from Human Plasma

Extracellular vesicles (EVs) are biological nanoparticles secreted by all cells for cellular communication and waste elimination. They participate in a vast range of functions by acting on and transferring their cargos to other cells in physiological and pathological conditions. Given their presence in biofluids, EVs represent an excellent resource for studying disease processes and can be considered a liquid biopsy for biomarker discovery. An attractive aspect of EV analysis is that they can be selected based on markers of their cell of origin, thus reflecting the environment of a specific tissue in their cargo. However, one of the major handicaps related to EV isolation methods is the lack of methodological consensuses and standardized protocols. Astrocytes are glial cells with essential roles in the brain. In neurodegenerative diseases, astrocyte reactivity may lead to altered EV cargo and aberrant cellular communication, facilitating/enhancing disease progression. Thus, analysis of astrocyte EVs may lead to the discovery of biomarkers and potential disease targets. This protocol describes a 2-step method of enrichment of astrocyte-derived EVs (ADEVs) from human plasma. First, EVs are enriched from defibrinated plasma via polymer-based precipitation. This is followed by enrichment of ADEVs through ACSA-1-based immunocapture with magnetic micro-beads, where resuspended EVs are loaded onto a column placed in a magnetic field. Magnetically labeled ACSA-1+ EVs are retained within the column, while other EVs flow through. Once the column is removed from the magnet, ADEVs are eluted and are ready for storage and analysis. To validate the enrichment of astrocyte markers, glial fibrillary acidic protein (GFAP), or other specific astrocytic markers of intracellular origin, can be measured in the eluate and compared with the flow-through. This protocol proposes an easy, time-efficient method to enrich ADEVs from plasma that can be used as a platform to examine astrocyte-relevant markers.

This protocol describes the enrichment of astrocyte-derived extracellular vesicles (ADEVs) from human plasma. It is based on the separation of EVs by polymer precipitation, followed by ACSA-1 based immunocapture of ADEVs. Analysis of ADEVs may offer clues to study changes in inflammatory pathways of living patients, non-invasively by liquid biopsy.

Extracellular vesicles (EVs) are biological nanoparticles secreted by all cells for cellular communication and waste elimination. They participate in a ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video