この作品は、健康補助金の国立研究所（R01-AI067395-01、R21-R022754-01、およびR21-I58002-01）によってサポートされていました。私たちは、原稿の重要な読書のためにC.ニーマイヤーに感謝します。

<ol>
	<li>Denny, 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+proteomes+of+human+parotid+and+ubmandibular/sublingual+gland+salivas+collected+as+the+ductal+secretions.">The proteomes of human parotid and ubmandibular/sublingual gland salivas collected as the ductal secretions.</a> J. Proteome Res. 7, 1994-2006 (2008).</li><li>Hu, S., Loo, J. A., Wong, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+saliva+proteome+analysis.">Human saliva proteome analysis.</a> Ann. N.Y. Acad. Sci. 1098, 323-329 (2007).</li><li>Ng, D. P., Koh, D., Choo, S. G., Ng, V., Fu, Q. <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+storage+conditions+on+the+extraction+of+PCR-quality+genomic+DNA+from+saliva.">Effect of storage conditions on the extraction of PCR-quality genomic DNA from saliva.</a> Clin. Chim. Acta. 343, 191-194 (2004).</li><li>Wade, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+oral-diffusion-sink+device+for+extended+sampling+of+multiple+steroid+hormones+from+saliva.">An oral-diffusion-sink device for extended sampling of multiple steroid hormones from saliva.</a> Clin. Chem. 38, 1878-1882 (1992).</li><li>Hu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+identification+of+proteins+in+human+salivary+proteome+by+liquid+chromatography/mass+spectrometry+and+two-dimensional+gel+electrophoresis-mass+spectrometry.">Large-scale identification of proteins in human salivary proteome by liquid chromatography/mass spectrometry and two-dimensional gel electrophoresis-mass spectrometry.</a> Proteomics. 5, 1714-1728 (2005).</li><li>Huang, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+proteomic+analysis+of+human+whole+saliva.">Comparative proteomic analysis of human whole saliva.</a> Arch. Oral Biol. 49, 951-962 (2004).</li><li>Guerrier, L., Lomas, L., Boschetti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+monobuffer+multidimensional+chromatography+for+high-throughput+proteome+fractionation.">A simplified monobuffer multidimensional chromatography for high-throughput proteome fractionation.</a> J Chromatogr. A. 1073, 25-33 (2005).</li><li>Huang, C. M., Wang, C. C., Kawai, M., Barnes, S., Elmets, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surfactant+sodium+lauryl+sulfate+enhances+skin+vaccination:+molecular+characterization+via+a+novel+technique+using+ultrafiltration+capillaries+and+mass+spectrometric+proteomics.">Surfactant sodium lauryl sulfate enhances skin vaccination: molecular characterization via a novel technique using ultrafiltration capillaries and mass spectrometric proteomics.</a> Mol. Cell Proteomics. 5, 523-532 (2006).</li><li>Huang, C. M., Wang, C. C., Kawai, M., Barnes, S., Elmets, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+protein+sampling+using+capillary+ultrafiltration+semi-permeable+hollow+fiber+and+protein+identification+via+mass+spectrometry-based+proteomics.">In vivo protein sampling using capillary ultrafiltration semi-permeable hollow fiber and protein identification via mass spectrometry-based proteomics.</a> J. Chromatogr. A. 1109, 144-151 (2006).</li><li>Huang, C. M., Wang, C. C., Barnes, S., Elmets, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+detection+of+secreted+proteins+from+wounded+skin+using+capillary+ultrafiltration+probes+and+mass+spectrometric+proteomics.">In vivo detection of secreted proteins from wounded skin using capillary ultrafiltration probes and mass spectrometric proteomics.</a> Proteomics. 6, 5805-5814 (2006).</li><li>Huang, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometric+proteomics+profiles+of+in+vivo+tumor+secretomes:+capillary+ultrafiltration+sampling+of+regressive+tumor+masses.">Mass spectrometric proteomics profiles of in vivo tumor secretomes: capillary ultrafiltration sampling of regressive tumor masses.</a> Proteomics. 6, 6107-6116 (2006).</li><li>Ahmed, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+approach+to+remove+albumin+for+the+proteomic+analysis+of+low+abundance+biomarkers+in+human+serum.">An approach to remove albumin for the proteomic analysis of low abundance biomarkers in human serum.</a> Proteomics. 3, 1980-1987 (2006).</li><li>Michishige, F. <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+saliva+collection+method+on+the+concentration+of+protein+components+in+saliva.">Effect of saliva collection method on the concentration of protein components in saliva.</a> J. Med. Invest. 53, 140-146 (2006).</li><li>Kruger, C., Breunig, U., Biskupek-Sigwart, J., Dorr, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Problems+with+salivary+17-hydroxyprogesterone+determinations+using+the+Salivette+device.">Problems with salivary 17-hydroxyprogesterone determinations using the Salivette device.</a> Eur. J. Clin. Chem. Clin. Biochem. 34, 926-929 (1996).</li><li>Luque-Garcia, J. L., Neubert, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+preparation+for+serum/plasma+profiling+and+biomarker+identification+by+mass+spectrometry.">Sample preparation for serum/plasma profiling and biomarker identification by mass spectrometry.</a> J. Chromatogr. A. 1153, 259-276 (2007).</li><li>Ramstrom, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+high-abundant+proteins+in+body+fluids+prior+to+liquid+chromatography+fourier+transform+ion+cyclotron+resonance+mass+spectrometry.">Depletion of high-abundant proteins in body fluids prior to liquid chromatography fourier transform ion cyclotron resonance mass spectrometry.</a> J. Proteome. Res. 4, 410-416 (2005).</li><li>Messana, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+human+salivary+basic+proline-rich+protein+complex+by+a+proteomic+approach.">Characterization of the human salivary basic proline-rich protein complex by a proteomic approach.</a> J. Proteome. Res. 3, 792-800 (2004).</li><li>Li, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possible+release+of+an+ArgGlyArgProGln+pentapeptide+with+innate+immunity+properties+from+acidic+proline-rich+proteins+by+proteolytic+activity+in+commensal+streptococcus+and+actinomyces+species.">Possible release of an ArgGlyArgProGln pentapeptide with innate immunity properties from acidic proline-rich proteins by proteolytic activity in commensal streptococcus and actinomyces species.</a> Infect. Immun. 68, 5425-5429 (2000).</li><li>Davtyan, T. K., Manukyan, H. A., Mkrtchyan, N. R., Avetisyan, S. A., Galoyan, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothalamic+proline-rich+polypeptide+is+a+regulator+of+oxidative+burst+in+human+neutrophils+and+monocytes.">Hypothalamic proline-rich polypeptide is a regulator of oxidative burst in human neutrophils and monocytes.</a> Neuroimmunomodulation. 12, 270-284 (2005).</li><li>Jonsson, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gln-Gly+cleavage:+correlation+between+collision-induced+dissociation+and+biological+degradation.">Gln-Gly cleavage: correlation between collision-induced dissociation and biological degradation.</a> J. Am. Soc. Mass Spectrom. 12, 337-342 (2001).</li><li>Gibbons, R. J., Hay, D. I., Schlesinger, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delineation+of+a+segment+of+adsorbed+salivary+acidic+proline-rich+proteins+which+promotes+adhesion+of+Streptococcus+gordonii+to+apatitic+surfaces.">Delineation of a segment of adsorbed salivary acidic proline-rich proteins which promotes adhesion of Streptococcus gordonii to apatitic surfaces.</a> Infect Immun. 59, 2948-2954 (1991).</li><li>Li, T., Johansson, I., Hay, D. I., Stromberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strains+of+Actinomyces+naeslundii+and+Actinomyces+viscosus+exhibit+structurally+variant+fimbrial+subunit+proteins+and+bind+to+different+peptide+motifs+in+salivary+proteins.">Strains of Actinomyces naeslundii and Actinomyces viscosus exhibit structurally variant fimbrial subunit proteins and bind to different peptide motifs in salivary proteins.</a> Infect Immun. 67, 2053-2059 (1999).</li><li>Hardt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+defining+the+human+parotid+gland+salivary+proteome+and+peptidome:+identification+and+characterization+using+2D+SDS-PAGE,+ultrafiltration,+HPLC,+and+mass+spectrometry.">Toward defining the human parotid gland salivary proteome and peptidome: identification and characterization using 2D SDS-PAGE, ultrafiltration, HPLC, and mass spectrometry.</a> Biochemistry. 44, 2885-2899 (2005).</li><li>Wilmarth, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+liquid+chromatography+study+of+the+human+whole+saliva+proteome.">Two-dimensional liquid chromatography study of the human whole saliva proteome.</a> J. Proteome Res. 3, 1017-1023 (2004).</li><li>Saitoh, E., Isemura, S., Sanada, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+amino+acid+sequence+of+a+basic+proline-rich+peptide,+P-D,+from+human+parotid+saliva.">Complete amino acid sequence of a basic proline-rich peptide, P-D, from human parotid saliva.</a> J. Biochem. 93, 495-502 (1983).</li><li>Slomiany, B. L., Piotrowski, J., Czajkowski, A., Shovlin, F. E., Slomiany, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+salivary+mucin+bacterial+aggregating+activity+with+caries+status.">Differential expression of salivary mucin bacterial aggregating activity with caries status.</a> Int. J. Biochem. 25, 935-940 (1993).</li><li>Juarez, Z. E., Stinson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+extracellular+protease+of+Streptococcus+gordonii+hydrolyzes+type+IV+collagen+and+collagen+analogues.">An extracellular protease of Streptococcus gordonii hydrolyzes type IV collagen and collagen analogues.</a> Infect Immun. 67, 271-278 (1999).</li><li>Lo, C. S., Hughes, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+protease+from+Streptococcus+oralis+C104.">Identification and characterization of a protease from Streptococcus oralis C104.</a> Oral Microbiol. Immunol. 11, 181-187 (1996).</li><li>Harrington, D. J., Russell, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterisation+of+two+extracellular+proteases+of+Streptococcus+mutans.">Identification and characterisation of two extracellular proteases of Streptococcus mutans.</a> FEMS Microbiol. Lett. 121, 237-241 (1994).</li><li>Huang, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+secretome+sampling+technology+for+proteomics.">In vivo secretome sampling technology for proteomics.</a> Proteomics Clin. Appl. 1, 953-962 (2007).</li><li>Skepo, M., Linse, P., Arnebrant, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coarse-grained+modeling+of+proline+rich+protein+1+(PRP-1)+in+bulk+solution+and+adsorbed+to+a+negatively+charged+surface.">Coarse-grained modeling of proline rich protein 1 (PRP-1) in bulk solution and adsorbed to a negatively charged surface.</a> J. Phys. Chem. B. 110, 12141-12148 (2006).</li><li>Losic, D., Rosengarten, G., Mitchell, J. G., Voelcker, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pore+architecture+of+diatom+frustules:+potential+nanostructured+membranes+for+molecular+and+particle+separations.">Pore architecture of diatom frustules: potential nanostructured membranes for molecular and particle separations.</a> J. Nanosci. Nanotechnol. 6, 982-989 (2006).</li><li>Linhares, M. C., Kissinger, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetic+monitoring+in+subcutaneous+tissue+using+in+vivo+capillary+ultrafiltration+probes.">Pharmacokinetic monitoring in subcutaneous tissue using in vivo capillary ultrafiltration probes.</a> Pharm Res. 10, 598-602 (1993).</li><li>Li, W., Hendrickson, C. L., Emmett, M. R., Marshall, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+intact+proteins+in+mixtures+by+alternated+capillary+liquid+chromatography+electrospray+ionization+and+LC+ESI+infrared+multiphoton+dissociation+Fourier+transform+ion+cyclotron+resonance+mass+spectrometry.">Identification of intact proteins in mixtures by alternated capillary liquid chromatography electrospray ionization and LC ESI infrared multiphoton dissociation Fourier transform ion cyclotron resonance mass spectrometry.</a> Anal. Chem. 71, 4397-4402 (1999).</li><li>Whitelegge, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-Sequence+Polymorphisms+and+Post-translational+Modifications+in+Proteins+from+Human+Saliva+using+Top-Down+Fourier-transform+Ion+Cyclotron+Resonance+Mass+Spectrometry.">Protein-Sequence Polymorphisms and Post-translational Modifications in Proteins from Human Saliva using Top-Down Fourier-transform Ion Cyclotron Resonance Mass Spectrometry.</a> Int. J. Mass Spectrom. 268, 190-197 (2007).</li><li>Castro, P., Tovar, J. A., Jaramillo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+of+Streptococcus+mutans+to+salivary+proteins+in+caries-free+and+caries-susceptible+individuals.">Adhesion of Streptococcus mutans to salivary proteins in caries-free and caries-susceptible individuals.</a> Acta Odontol. Latinoam. 19, 59-66 (2006).</li><li>Challacombe, S. J., Sweet, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+mucosal+immunity+and+HIV+infection:+current+status.">Oral mucosal immunity and HIV infection: current status.</a> Oral Dis. 8, 55-62 (2002).</li><li>Jensen, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salivary+acidic+proline-rich+proteins+in+rheumatoid+arthritis.">Salivary acidic proline-rich proteins in rheumatoid arthritis.</a> Ann. N.Y. Acad. Sci. 842, 209-211 (1998).</li></ol>

利害の衝突が宣言されません。

我々は、多くのペプチドフラグメントは、人間の消化唾液中に存在することを発見した。これらのペプチド断片は、プロリンリッチタンパク質、アクチン、α-アミラーゼ、α1グロビン、βグロビン、histain 1、ケラチン1、ムチン7、高分子免疫グロブリン受容体、satherin、S100A9の様々なフォームからの誘導体である。未定の切断部位を有するペプチドの生産に貢献する多くの要因があるかもしれ?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>ポリエーテルスルホン膜</td><td>ポール·コーポレーション</td><td></td><td> 30kDaのMWCO </td></tr><tr><td>テフロンフッ素化エチレンプロピレン管</td><td>アップチャーチ科学</td><td></td><td></td></tr><tr><td>ブルーデキストラン</td><td>シグマ</td><td></td><td></td></tr><tr><td>ナノLCシステム</td><td> Eksigent </td><td></td><td></td></tr><tr><td> C18トラップカラム</td><td>アジレント</td><td> 5065-9913 </td><td></td></tr><tr><td> LTQリニアイオントラップ型質量分析計</td><td>サーモフィッシャー</td><td></td><td></td></tr><tr><td>魔術師2 </td><td> SAGE-N ReseaRchの</td><td></td><td></td></tr><tr><td>アセトニトリル0.1％ギ酸</td><td> JTベーカー</td><td> 9832から03 </td><td> LC / MSグレード</td></tr><tr><td>水0.1％ギ酸</td><td> JTベーカー</td><td> 9834から03 </td><td> LC / MSグレード</td></tr></tbody></table>

sampling human indigenous saliva peptidome using a lollipop-like ultrafiltration probe: simplify and enhance peptide detection for clinical mass spectrometry

ヒト唾液プロテオームとペプチドーが1-2を明らかにされているが、それらはすぎなかったトリプシン唾液タンパク質の消化物から同定された。外因性の酵素による事前消化せずにヒト唾液の先住民族ペプチドーの同定は、ヒト唾液でネイティブペプチドが疾患の進行を予測し、治療効果を監視し、病気を診断するための潜在的な値を提供するので、必須となります。適切なサンプリングは、人間の土着の唾液ペプチドーの識別の強化のための重要なステップです。 3から4の残骸を削除するには、遠心分離を含むヒト唾液をサンプリングする従来の方法は時間がかかりすぎるの臨床使用のために適用可能であることかもしれません。さらに、遠心分離による瓦礫撤去は、感染した病原体のほとんどをきれいにし、多くの場合、低濃度のペプチドーの識別を妨げる高濃度タンパク質を除去することができない場合があります。 PRI、従来のプロテオミクス的手法marilyゲル内消化との結合の二次元ゲル電気泳動（2-DE）ゲルは多くの唾液タンパク質の5-6を識別することができる利用しています。しかし、このアプローチは、一般的に低濃度のペプチド/タンパク質を検出するのに十分な小文字は区別されません。液体クロマトグラフィー - 質量分析（LC-MS）ベースのプロテオミクス前の2-DE分離することなくタンパク質を識別することができる代替手段です。このアプローチは、より高い感度を提供していますが、それは一般的に臨床使用が困難になってトリプシンと以前のサンプルプレ分画7とプレ消化を必要とします。 サンプル調製のために質量分析の妨害を回避するために、我々は、キャピラリ限外濾過（CUF）プローブ8から11と呼ばれる技術を開発しました。我々の研究室からのデータは、CUFプローブは、ダイナミックかつ低侵襲な方法8の動物の様々な微小環境から、生体内でタンパク質を捕捉することが可能であることを実証-11。負圧は、サンプル収集中に撤退単に注射器によって作成されているので全く遠心分離は必要ありません。 LC-MSを組み合わせたCUFプローブが成功したトリプシン消化し ​​たタンパク質の8-11を同定した。本研究では、簡単に人間の口腔内に収まることができロリポップのような限外濾過（LLUF）プローブを作成することにより、限外ろ過サンプリング技術をアップグレードしました。トリプシン消化せずにLC-MSによる直接分析では、国産ヒト唾液は様々なタンパク質に由来する多くのペプチド断片が含まれていることを示した。 LLUFプローブと唾液をサンプリングする遠心分離を避けますが、効果的に多くの大規模、高濃度タンパク質を除去。私たちの質量分析の結果は、多くの低濃度のペプチドはLLUFプローブで大きなタンパク質をフィルタリングした後に検出されなったことを示す。低濃度の唾液ペプチドの検出は、クロマトグラフィーを持つ複数のステップのサンプルの分離独立していた。臨床応用のために、LLUFプローブが組み込まれてLC-MSとdが潜在的に唾液から疾患の進行を監視するために、将来的に使用することができます。

Watch this Scientific Journal Video about Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry 

Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry

将来の臨床応用のための唾液採取を考慮して、ロリポップのような限外濾過（LLUF）プローブは、ヒトの口腔内に収まるように作製した。ナノLC-LTQ質量分析法による消化唾液の直接分析は、大規模なタンパク質や高濃度タンパク質を除去し、低豊富なペプチドは多くの検出可能なようにするLLUFプローブの能力を実証した。

ロリポップライク限外プローブを用いたヒト先住民唾液ペプチドーをサンプリング：臨床質量分析のためのペプチドの検出を簡素化し、強化

Although human saliva proteome and peptidome have been revealed 1-2 they were majorly identified from tryptic digests of saliva proteins. Identification ...

sampling-human-indigenous-saliva-peptidome-using-lollipop-like

Research

JoVE Journal

Medicine

11.4K ビュー.  Sanford-Burnham Medical Research Institute. 将来の臨床応用のための唾液採取を考慮して、ロリポップのような限外濾過（LLUF）プローブは、ヒトの口腔内に収まるように作製した。ナノLC-LTQ質量分析法による消化唾液の直接分析は、大規模なタンパク質や高濃度タンパク質を除去し、低豊富なペプチドは多くの検出可能なようにするLLUFプローブの能力を実証した。

ビデオ: Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry

Using Visual and Narrative Methods to Achieve Fair Process in Clinical Care

The Institute of Medicine has targeted patient-centeredness as an important area of quality improvement. A major dimension of patient-centeredness is respect for patient's values, preferences, and expressed needs. Yet specific approaches to gaining this understanding and translating it to quality care in the clinical setting are lacking. From a patient perspective quality is not a simple concept but is best understood in terms of five dimensions: technical outcomes; decision-making efficiency; amenities and convenience; information and emotional support; and overall patient satisfaction. Failure to consider quality from this five-pronged perspective results in a focus on medical outcomes, without considering the processes central to quality from the patient's perspective and vital to achieving good outcomes. In this paper, we argue for applying the concept of fair process in clinical settings. Fair process involves using a collaborative approach to exploring diagnostic issues and treatments with patients, explaining the rationale for decisions, setting expectations about roles and responsibilities, and implementing a core plan and ongoing evaluation. Fair process opens the door to bringing patient expertise into the clinical setting and the work of developing health care goals and strategies. This paper provides a step by step illustration of an innovative visual approach, called photovoice or photo-elicitation, to achieve fair process in clinical work with acquired brain injury survivors and others living with chronic health conditions. Applying this visual tool and methodology in the clinical setting will enhance patient-provider communication; engage patients as partners in identifying challenges, strengths, goals, and strategies; and support evaluation of progress over time. Asking patients to bring visuals of their lives into the clinical interaction can help to illuminate gaps in clinical knowledge, forge better therapeutic relationships with patients living with chronic conditions such as brain injury, and identify patient-centered goals and possibilities for healing. The process illustrated here can be used by clinicians, (primary care physicians, rehabilitation therapists, neurologists, neuropsychologists, psychologists, and others) working with people living with chronic conditions such as acquired brain injury, mental illness, physical disabilities, HIV/AIDS, substance abuse, or post-traumatic stress, and by leaders of support groups for the types of patients described above and their family members or caregivers.

This paper illustrates an innovative visual approach (photovoice or photo-elicitation) to achieve fair process in clinical care for patients living with chronic health conditions, illuminate gaps in clinical knowledge, forge better therapeutic relationships, and identify patient-centered goals and possibilities for healing.

臨床ケアのフ​​ェアプロセスを達成するために、Visualと物語メソッドの使用

The Institute of Medicine has targeted patient-centeredness as an important area of quality improvement. A major dimension of patient-centeredness is ...

医学

The &alpha;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time and temperature restrictions, technical sophistication, and cost of reagents, in particular monoclonal antibodies to measure CD4 on blood cells, the only currently acceptable method. A commonly used cost-saving and time-saving laboratory strategy is to calculate, rather than measure certain blood values. For example, LDL levels are calculated using the measured levels of total cholesterol, HDL, and triglycerides1. Thus, identification of cell-free correlates that directly regulate the number of CD4+ T cells could provide an accurate method for calculating CD4 counts due to the physiological relevance of the correlates.
The number of stem cells that enter blood and are destined to become circulating CD4+ T cells is determined by the chemokine CXCL12 and its receptor CXCR4 due to their influence on locomotion2. The process of stem cell locomotion into blood is additionally regulated by cell surface human leukocyte elastase (HLECS) and the HLECS-reactive active &alpha;1proteinase inhibitor (&alpha;1PI, &alpha;1antitrypsin, SerpinA1)3. In HIV-1 disease, &alpha;1PI is inactivated due to disease processes 4. In the early asymptomatic categories of HIV-1 disease, active &alpha;1PI was found to be below normal in 100% of untreated HIV-1 patients (median=12 &mu;M, and to achieve normal levels during the symptomatic categories4, 5. This pattern has been attributed to immune inactivation, not to insufficient synthesis, proteolytic inactivation, or oxygenation. We observed that in HIV-1 subjects with &gt;220 CD4 cells/&mu;l, CD4 counts were correlated with serum levels of active &alpha;1PI (r2=0.93, p&lt;0.0001, n=26) and inactive &alpha;1PI (r2=0.91, p&lt;0.0001, n=26) 5. Administration of &alpha;1PI to HIV-1 infected and uninfected subjects resulted in dramatic increases in CD4 counts suggesting &alpha;1PI participates in regulating the number of CD4+ T cells in blood 3.
With stimulation, whole saliva contains sufficient serous exudate (plasma containing proteinaceous material that passes through blood vessel walls into saliva) to allow measurement of active &alpha;1PI and the correlation of this measurement is evidence that it is an accurate method for calculating CD4 counts. Briefly, sialogogues such as chewing gum or citric acid stimulate the exudation of serum into whole mouth saliva. After stimulating serum exudation, the activity of serum &alpha;1PI in saliva is measured by its capacity to inhibit elastase activity. Porcine pancreatic elastase (PPE) is a readily available inexpensive source of elastase. PPE binds to &alpha;1PI forming a one-to-one complex that prevents PPE from cleaving its specific substrates, one of which is the colorimetric peptide, succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA3NA). Incubating saliva with a saturating concentration of PPE for 10 min at room temperature allows the binding of PPE to all the active &alpha;1PI in saliva. The resulting inhibition of PPE by active &alpha;1PI can be measured by adding the PPE substrate SA3NA. (Figure 1). Although CD4 counts are measured in terms of blood volume (CD4 cells/&mu;l), the concentration of &alpha;1PI in saliva is related to the concentration of serum in saliva, not to volume of saliva since volume can vary considerably during the day and person to person6. However, virtually all the protein in saliva is due to serum content, and the protein content of saliva is measurable7. Thus, active &alpha;1PI in saliva is calculated as a ratio to saliva protein content and is termed the &alpha;1PI Index. Results presented herein demonstrate that the &alpha;1PI Index provides an accurate and precise physiologic method for calculating CD4 counts.

The &amp;#945;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

A CD4 enumeration method, the &#x03B1;-test, is described which uses whole saliva to provide rapid and accurate CD4 counts. The &alpha;-test costs pennies and eliminates the need for technical training, costly reagents such as monoclonal antibodies, instrumentation, refrigeration, transport of samples, as well as collection and handling of blood.

α-テスト：全唾液を用いた迅速な無細胞CD4列挙

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time ...

Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers

Salivary measures have emerged in bio-behavioral research that are easy-to-collect, minimally invasive, and relatively inexpensive biologic markers of stress. This article we present the steps for collection and analysis of two salivary assays in research with frail, community residing older adults-salivary cortisol and salivary alpha amylase. The field of salivary bioscience is rapidly advancing and the purpose of this presentation is to provide an update on the developments for investigators interested in integrating these measures into research on aging. Strategies are presented for instructing family caregivers in collecting saliva in the home, and for conducting laboratory analyses of salivary analytes that have demonstrated feasibility, high compliance, and yield quality specimens. The protocol for sample collection includes: (1) consistent use of collection materials; (2) standardized methods that promote adherence and minimize subject burden; and (3) procedures for controlling certain confounding agents. We also provide strategies for laboratory analyses include: (1) saliva handling and processing; (2) salivary cortisol and salivary alpha amylase assay procedures; and (3) analytic considerations.

We demonstrate: (1) procedures for collection of salivary samples in cognitive impaired older adults by family caregivers in the home setting, (2) procedures for measuring stress activity via salivary cortisol and alpha amylase, and (3) representative profiles. Protocols that allow researchers to study stress-linked processes advance our understanding of biological sensitivity and susceptibility.

唾液を収集し、家族介護者を経由して高齢者を居住する虚弱コミュニティ唾液コルチゾールおよびα-アミラーゼの測定

Salivary measures have emerged in bio-behavioral research  that are easy-to-collect, minimally invasive, and relatively inexpensive  biologic markers of ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

液体および固形腫瘍由来の癌細胞のプロテオームの定量的質量分析プロファイリング

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

ヒト前立腺癌の前臨床同所性マウスモデル

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

A Detailed Protocol for Perspiration Monitoring Using a Novel, Small, Wireless Device

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the patients are unaware of such disorders or are having difficulty expressing their symptoms. Until now, several devices for perspiration monitoring have been developed; however, such devices tend to have a relatively large exterior, considerable power consumption, and/or less sensitivity.
Recently, we developed a small, wireless device for perspiration monitoring. The device consists of a temperature/relative humidity (T/RH) sensor, battery-driven small data logger, and silica gel as a desiccant in a small cylindrical exterior. The T/RH sensor is placed between the detection windows (through which the water vapor from the skin enters) and the silica gel. The underlying principle of the perspiration monitoring device is based on Fick&#39;s law of diffusion, which means that water vapor flux from the skin to the silica gel (i.e.&#160;transepidermal water loss and perspiration) can be captured by change in humidity at the T/RH sensor. In addition, a baseline subtraction method was adopted to distinguish perspiration and transepidermal water loss.
As shown in the previous report, the developed device can monitor the perspiration at any sites of the body in an easy, wireless manner. However, detailed methods of how to use the device have not been disclosed yet. In this article, therefore, we would like to show the point-by-point tutorials of how to use the device for perspiration monitoring, by showing the sympathetic activity test with the sympathetic skin response monitoring as an example.

Recently, we developed a small wireless device for perspiration monitoring. In this article, we present detailed protocols on how to use the device for perspiration monitoring with an example of the sympathetic activity test.

汗は小説、小型、ワイヤレスデバイスを使用した監視するための詳細なプロトコル

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the ...

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular Stromal Cells (PSCs) share characteristics with bone marrow MSCs (bmMSCs). However, they also possess, most likely due to local imprinting, tissue-specific properties and play a role in local tissue homeostasis. This tissue specificity may result in tissue specific repair, also within the human kidney. We previously showed that human kidney PSCs (kPSCs) have enhanced kidney epithelial wound healing whereas bmMSCs did not have this potential. Moreover, kPSCs can ameliorate kidney injury in vivo. Therefore, kPSCs constitute an interesting source for cell therapy, particularly for kidney diseases and renal transplantation. Here we show the detailed isolation and culture method for kPSCs from transplant-grade human kidneys based on whole-organ perfusion of digestive enzymes via the renal artery and enrichment for the perivascular marker NG2. In this way, large cell quantities can be obtained that are suitable for cellular therapy.

Here we present a novel clinical grade isolation and culture method for kidney Perivascular Stromal Cells (kPSCs) based on whole organ perfusion with digestive enzymes and NG2-cell enrichment. With this method, it is possible to acquire sufficient cell numbers for cellular therapy.

ひと腎臓血管周囲間質細胞の新規臨床等級分離法

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

In VitroとIn Vivo検出、ひと細胞、線虫、マウス Mitophagy の

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Surgical Training for the Implantation of Neocortical Microelectrode Arrays Using a Formaldehyde-fixed Human Cadaver Model

This protocol describes a procedure to assist surgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain. Recent technological progress has enabled the fabrication of microelectrode arrays that allow recording the activity of multiple individual neurons in the neocortex of the human brain. These arrays have the potential to bring unique insight onto the neuronal correlates of cerebral function in health and disease. Furthermore, the identification and decoding of volitional neuronal activity opens the possibility to establish brain-computer interfaces, and thus might help restore lost neurological functions. The implantation of neocortical microelectrode arrays is an invasive procedure requiring a supra-centimetric craniotomy and the exposure of the cortical surface; thus, the procedure must be performed by an adequately trained neurosurgeon. In order to provide an opportunity for surgical training, we designed a procedure based on a human cadaver model. The use of a formaldehyde-fixed human cadaver bypasses the practical, ethical and financial difficulties of surgical practice on animals (especially non-human primates) while preserving the macroscopic structure of the head, skull, meninges and cerebral surface and allowing realistic, operating room-like positioning and instrumentation. Furthermore, the use of a human cadaver is closer to clinical daily practice than any non-human model. The major drawbacks of the cadaveric simulation are the absence of cerebral pulsation and of blood and cerebrospinal fluid circulation. We suggest that a formaldehyde-fixed human cadaver model is an adequate, practical and cost-effective approach to ensure proper surgical training before implanting microelectrode arrays in the living human neocortex.

We designed a procedure in which a formaldehyde-fixed human cadaver is used to assist neurosurgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain.

人間の死体をホルマリン固定モデルを用いた皮質微小電極アレイの注入の手術トレーニング

This protocol describes a procedure to assist surgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain. ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

サンプリング戦略とブタの生物医学的モデルからバイオバンク組織サンプルの処理

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...