ISSN 1007-7626 CN 11-3870 / Q http/ / cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology 2012 1 281 86 ~ 92 1 2 1 1 1 1 1 3* 1 201508 2 450052 3 410078 Tricine- SDS-PAGE MRM ESAT-6 ESAT-6 MRM Tricine-SDS-PAGE MRM 0 999 MRM 0 995 MRM Q5-33 Quantification of Target Peptides or Proteins by Liquid Chromatography- Mass Spectrometry with Multiple Reaction Monitoring 1 LIU Yong-Fu 2 JIA Xiao-Fang 1 TENG Zhen-Lin 1 YIN Lin 1 LIU Bao-Chi 1 1 ZHANG Li-Jun 3* 1 Shanghai Public Health Clinical CenterShanghai 201508China 2 First Affiliated Hospital of Zhengzhou UniversityZhengzhou 450052China 3 Institute of Clinical PharmacologyCentral South UniversityChangsha 410078China Abstract This study was to establish a modified plasma peptide extraction method and to develop a mass spectrometry quantification method for target peptides or proteins Three techniques of ultrafiltration organic solvent extraction and solid phase extraction were used to extract peptides from plasma samples Tricine-SDS-PAGE was used to determine the extraction efficiency Peptide and protein quantification were completed by liquid chromatography combined with mass spectrometry in multiple reaction monitoring MRMmode The Tricine-SDS-PAGE results showed that acetonitrile ACNprecipitation was the most efficient approach for low molecular plasma peptides enrichment besides for excluding the high molecular protein contaminations The correlation coefficients of the standard curves were 0 995 and 0 999 in the quantification of peptide or protein standards by liquid chromatography-mass spectrometry LC-MS respectively In conclusionwe successfully established a simple and efficient plasma peptide extraction method and a subsequent LC-MS MRM method for peptide and protein quantifications These 2011-09-06 2011-10-28 No 20100471000 No 201104484 No 20100031 * Tel021-37990333-5368E-mailzhanglijun1221@ 163 com ReceivedSeptember 62011AcceptedOctober 282011 Supported by Postdoctoral Science Foundation of China No 20100471000 No 201104484 Wang Bao-En Liver Fibrosis Research Foundation from Chinese Foundation for Hepatitis Prevention and Control No 20100031 * Corresponding author Tel021-37990333-5368E-mailzhanglijun1221@ 163 com
1 87 methods might be applied to the quantification of target peptides or proteins in complex samples Key words peptide protein multiple reaction monitoring MRM quantification liquid chromatography-mass spectrometrylc-ms LABCONCO Fig 1 Overall workflow for developing peptide and Ultimate 3000 C18 C18 protein quantification method Endogenous peptides in rat plasma were extracted by ultrafiltrationorganic solvent HCT extraction or SPE treatment Tricine-SDS-PAGE was then used LC-20AD to verify the peptide extraction efficiency Moreovera API3200 standard peptide ESAT-6 was used to develop a MRM peptide Eppendorf quantification method using API3200 mass spectrometryand BACKMAN COULTER BIO- this method was further applied to standard protein BSA RAD SDS-PAGE quantification GE Image scanner JA2003A 1 2 1 SD Tricine- SDS-PAGE multiple reaction monitoringmrm 23 4 ~ 6 7 1 1 1 Sartorius BP211D WATERS C18 SPE Millipore ultracel YM-10 10 kd N N - USB 50 μl 100 APs NNN N - μl = 1 2 500 μl = TEMED IAA AMRESCO Tris acetonitrileacn MeOH Merck Fig 1 1 2 1 0 1% 1 10 0 1% TFA 4 GE 30 min 4 12 000 r / min 20 min 20 min FA Fluka SDS Sigma HAc 100% 1 2 2 2 15% 60%
88 28 12 Waters C18 kd 64 ~ 76 SPE 500 μl SPE 1 2 500 μl 75% ACN 0 1% FA 5 μl 6 25 12 5 25 50 100 200 ng / μl SPE 1 3 500 μl 2% ESAT-6 45 μl ACN 0 1% FA SPE 0 625 1 25 2 5 5 10 20 ng / μl ESAT-6 42 50 μl 450 μl 2% 12 0 1% ACN 0 1% FA 2 100 μl 2% 0 1% SPE SPE 10 μl API3200 SPE 5 500 μl 2% ACN 0 1% FA 735 5 /389 3 735 5 /460 3 735 5 /524 4 6 2 SPE 1 3 3 MRM 500 μl 15% 60% ACN 1 MRM Fig 2 1 0 375 0 75 1 5 2 25 3 4 5 6 μg BSA11 5% SDS-PAGE 1 2 3 50 μl 450 μl G-205 BSA 10% ACN 4 30 min Trypsin 4 3 000 g 30 min BSA BSA 1 2 4 Tricine-SDS-PAGE Ultimate 3000 Bruker HCT 9 20 μl 12% SDS 6% β- 30% 150 mmol / L Tris-HCl ph mascot peptide ion score 39 2 7 0 Tricine-SDS-PAGE4% C M NG N Q N E3 10% 16% 8 1 3 MRM 1 3 1 MRM P1 P26 1-25 7 LC-20AD NCBI expacy protein blast API3200 A0 1% B80% unique peptide 0 1% 1 0 mm 150 mm 300 C18 0 06 ml / min 0 ~ 12 4 min 5% ~ 62 5% B12 1 ~ 20 min 62 5% B20 1 ~ 25 min 100% B25 1 ~ 50 min 5% B API3200 P C P P1 P2 Pro N CUR10IS5500TEM300GS150GS250 Y BSA iheoncad6dp50ep80ce35cxp 8 Applied Biosystems API3200 BSA MRM 4 735 5 /215 5 1 R K4 5 C Pro 1 HCT 4 Y m / z 8002 LC-20AD > 1 000 1 3 2 MRM MRM Fig 1 2 API3200 1 ELNNALQNLARTI ESAT- 2 1 6 6
1 89 Tricine-SDS- 2 60% PAGE Fig 2 15 kd 2 3 Tricine-SDS-PAGE 2 1% Fig 2 Tricine-SDS-PAGE of plasma peptide samples extracted by different methods Five methods were used to extract endogenous peptides from rat plasma The subsequent peptide samples were then subjected to Tricine- SDS-PAGE separation and stained by Coomassie brilliant blue G-250 M Marker10 ~ 50 kda Ultrafiltration B 1 10 ACN 0 1% FA precipitationc 1 2 ACN 0 1% FA precipitation D 60% ACN elution SPE extractione 15% ACN elution SPE extraction 2 2 ESAT-6 MRM ESAT-6 ESAT-6 0 625 ~ 20 ng /μl r = 0 999 3 Fig 3 ESAT-6 4 735 5 /215 5735 5 /389 3735 5 /460 3 735 5 /524 4 MRM 735 5 /215 3 2 3 0 375-6 μg BSA SDS-PAGE Fig 4A BSA API3200 Fig 2 1 2 MRM 1% BSA 15 kd MRM BSA 10 BSA 0 375 ~ 6 μg Tricine- SDS-PAGE r = 0 994 9 Fig 3 MRM detection of a standard peptide ESAT-6 by API3200 mass spectrometry 5 μl of 6 2512 52550 100 and 200 ng / μl ESAT-6 were added into 45 μl plasma from health donor The peptide samples were extracted by adding 2 volumes of ACN 0 1% FA and analyzed by API3200 mass spectrometry using MRM method A MRM spectra of 4 transitions from ESAT-6735 5 /215 5735 5 /389 3735 5 /460 3 and 735 5 /524 4 BThe standard curve for ESAT-6 transitions 735 5 /215 3 in 0 625-20 ng / μl The correlation coefficient of ESAT-6 quantification standard curve was 0 9949
90 28 Fig 4B Table 1 BSA Fig 4 Standard curve for BSA MRM quantification ASDS-PAGE for BSA 0 3750 751 52 2534 5 and 6 μg BSA lane 123456 and 7respectively were subjected to 11 5% SDS-PAGE and then the gel was stained by Coomassie brilliant blue G-250 M represented for Marker The molecular mass of maker was showed on the left of the gel BThe standard curve for BSA quantification by API3200 mass spectrometry The BSA bands from SDS- PAGE were cut out and lysised by trypsinthen analyzed by mass spectrometry using MRM method The correlation coefficient of standard curve was 0 9949 Table 1 Peptides used for BSA quantification and the correlated precursor and fragment ions Peptide sequence Precursor ionsm / z Fragment ionsm / z KQTALVELLK 571 9 260 2y2 373 3y3 LVNELTEFAK 582 4 218 1y2 365 2y3 LGEYGFQNALIVR 740 5 274 2y2 387 3y3 DAFLGSFLYEYSR 785 0 262 2y2 425 2y3 LVVSTQTALA 501 8 203 1y2 11 12 ~ 14 1516 1718 19 Tricine-SDS-PAGE 3 1 2 0 1% 15 kd 12 0 1% Elisa 3 MRM 10 2001 Elisa MRM
1 91 biomarker discoveryj MRM MRM-MS Q1 Anal Chem201183 17 6834-6841 Q2 Q3 application to a pharmacokinetic study in rat plasmaj - Biomed Anal201156 3 623-626 MRM 300 - J J Proteome Res20109 9 4346-4355 MRM-MS LC-20D 8 1860-1877 API3200 6 MRM J SDS-PAGE proteome researchj Chem Life 200828 2 210-213 API3200 biomarkers for lung adenocarcinomaj 4 e18567 8 Schgger H Tricine-SDS-PAGEJ MRM 16-22 glycoproteomic analysisj 3437-3445 J 217 11Jiang XYe MZou H Proteomics20088 4 686-705 SDS-PAGE MRM 2 571-581 MRM Fourier transform mass spectrometerj 1120 1-2 173-184 References 1 Petricoin E FBelluco CAraujo R Pet al The blood peptidomea higher dimension of information content for cancer 967 Nat Rev Cancer20066 12 961-2 Liu YUboh C ESoma L Ret al Efficient use of retention time for the analysis of 302 drugs in equine plasma by liquid chromatography-ms / MS with scheduled multiple reaction monitoring and instant library searching for doping controlj 3 Lee J HLim HKim D Get al Quantification of oltipraz using liquid chromatography-tandem mass spectrometry and its J Pharm 4 Ang C SNice E C Targeted in-gel MRMa hypothesis driven approach for colorectal cancer biomarker discovery in human feces 5 Kuzyk M ASmith DYang Jet al Multiple reaction monitoring-basedmultiplexedabsolute quantitation of 45 proteins in human plasmaj Mol Cell Proteomics20098 MRM Zhao YanYing Wan-TaoQian Xiao-Hong Application of mass spectrometry MRM technology in 7 Ueda KSaichi NTakami Set al A comprehensive peptidome profiling technology for the identification of early detection PLoS One20116 Nat Protoc20061 1 9 Zhang LJia XZhang Xet al Alpha-1 antitrypsin variants in plasma from HIV-infected patients revealed by proteomic and Electrophoresis201031 20 10Schulz-Knappe PZucht H DHeine Get al Peptidomicsthe comprehensive analysis of peptides in complex biological mixtures Comb Chem High Throughput Screen20014 2 207- Technologies and methods for sample pretreatment in efficient proteome and peptidome analysisj 12Aristoteli L PMolloy M PBaker M S Evaluation of endogenous plasma peptide extraction methods for mass spectrometric biomarker discoveryj J Proteome Res20076 13Zheng XBaker HHancock W Analysis of the low molecular weight serum peptidome using ultrafiltration and a hybrid ion trap- J Chromatogr A2006 14Zougman APilch BPodtelejnikov Aet al Integrated analysis of the cerebrospinal fluid peptidome and proteome J J Proteome Res20087 1 386-399 15Fiedler G MBaumann SLeichtle Aet al Standardized
92 28 peptidome profiling of human urine by magnetic bead separation and matrix-assisted laser desorption / ionization time-of-flight mass spectrometryj Clin Chem200753 3 421-428 16Villanueva JPhilip JEntenberg Det al Serum peptide profiling by magnetic particle-assistedautomated sample processing and MALDI-TOF mass spectrometryj Anal Chem 200476 6 1560-1570 17Tucholska MScozzaro SWilliams Det al Endogenous peptides from biophysical and biochemical fractionation of serum analyzed by matrix-assisted laser desorption / ionization and electrospray ionization hybrid quadrupole time-of-flightj Anal Biochem 2007 3702 228-245 18Chertov OBiragyn AKwak L Wet al Organic solvent extraction of proteins and peptides from serum as an effective sample preparation for detection and identification of biomarkers by mass spectrometryj Proteomics20044 4 1195-1203 19Bertile FRobert FDelval-Dubois Vet al Endogenous plasma peptide detection and identification in the rat by a combination of fractionation methods and mass spectrometry J Biomark Insights20072385-401