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Second Edition, Version 2.03

April 2017

PlasmaDive™ Mini

Human Plasma Assay Panel for MRM or PRM analysis

MANUAL

Содержание PlasmaDive Mini

Страница 1: ...Second Edition Version 2 03 April 2017 Biognosys AG Switzerland PlasmaDive Mini Human Plasma Assay Panel for MRM or PRM analysis MANUAL ...

Страница 2: ... 4 Technical Assistance 4 Safety Information 4 Introduction PlasmaDive Mini Assay Panel at a Glance 5 Important Notes before Start 7 Sample Requirements 7 SpectroDive Software and PlasmaDive Mini Panel Plug in 8 Additionally Required Laboratory Equipment and Consumables 9 Additionally Required Reagents Solvents and Solutions 9 Sample Preparation Procedure LC MRM PRM Analysis 10 Report Columns Desc...

Страница 3: ...e upon request at support biognosys com PlasmaDive Mini Panel Manual Available at www biognosys com shop plasmadive mini Storage and Quality Control of PlasmaDive Mini Kit Immediately after receiving the kit store Reference Peptide Mix at 20 C Reduction Stock Solution and Alkylation Solution at 4 C and protected from light All other components should be stored dry at room temperature 15 25 C In ac...

Страница 4: ...tists with extensive practical and theoretical expertise in proteomic technologies and bioinformatics If you have any questions or experience any difficulties with PlasmaDive Mini Assay Panel please do not hesitate to contact us at support biognosys com call 41 44 738 20 40 or visit www biognosys com shop plasmadive mini Safety Information When working with chemicals always wear a suitable lab coa...

Страница 5: ...ini Assay Panel optimally combines 100 peptide MRM or PRM assays in one scheduled multiplexed method Each peptide is representative of a human plasma protein Table 1 The PlasmaDive Mini workflow requires as little as 10 µl of plasma and 48 hours to provide actionable clinically relevant data Table 1 List of proteins quantified with PlasmaDive Mini Assay Panel UniPot ID Entry Name Protein Name P027...

Страница 6: ...nent C8 alpha chain P02748 CO9_HUMAN Complement component C9 P02775 CXCL7_HUMAN Platelet basic protein C X C motif chemokine 7 P00488 F13A_HUMAN Coagulation factor XIII A chain P05160 F13B_HUMAN Coagulation factor XIII B chain P00742 FA10_HUMAN Coagulation factor X P00740 FA9_HUMAN Coagulation factor IX P23142 FBLN1_HUMAN Fibulin 1 P02765 FETUA_HUMAN Alpha 2 HS glycoprotein Q9UGM5 FETUB_HUMAN Fetu...

Страница 7: ...n P P04278 SHBG_HUMAN Sex hormone binding globulin P05452 TETN_HUMAN Tetranectin P05543 THBG_HUMAN Thyroxine binding globulin Serpin A7 P00734 THRB_HUMAN Prothrombin P02787 TRFE_HUMAN Serotransferrin P02766 TTHY_HUMAN Transthyretin P02774 VTDB_HUMAN Vitamin D binding protein P04004 VTNC_HUMAN Vitronectin P04275 VWF_HUMAN von Willebrand factor P25311 ZA2G_HUMAN Zinc alpha 2 glycoprotein Important N...

Страница 8: ... higher 4GB RAM 50GB free hard disc space software NET 4 5 SpectroDive Installation Close all running programs Open the SpectroDive installer administrator rights are required and follow the wizard instructions Carefully read through the license agreement Choose the location for installation Check settings and proceed with installation A confirmation message will appear after successful installati...

Страница 9: ...t 37 C Centrifuge with cooling option 4 C Vacuum concentrator LC MS vials Additionally Required Reagents Solvents and Solutions Sequencing grade modified trypsin stock solution at 0 4 µg µl recommended Promega Product Catalog V5113 Note 1 Solution can be stored at room temperature for up to one year Note 2 Use a glass syringe to pipette strong acidic solutions like concentrated TFA Note 3 Use 10 v...

Страница 10: ...o a new set of 1 5 ml tubes 5 Add 10 µl of a plasma sample in each tube Note 6 6 Gently shake the samples on thermomixer for 1 min at room temperature 7 Briefly spin down to collect all liquid at the bottom of the tubes 8 Incubate the samples at 37 C 600 rpm on thermomixer for 30 min 9 Let the samples cool to room temperature while preparing the Alkylation Solution 9 1 Fill up Alkylation Solution ...

Страница 11: ...using a single channel pipette 20 Gently shake the samples on thermomixer for 1 min at room temperature 21 Briefly spin down to collect all liquid at the bottom of the tubes 22 Incubate the samples at 37 C 600 rpm in the thermomixer for 3 hours 23 Acidify samples by adding 20 µl sample of 10 v v TFA solution using a single channel pipette Note 9 24 Check pH to be below 2 in a few samples by pipett...

Страница 12: ... g for 1 min and discard the flow through Note 10 37 Repeat step 36 two more times Elution and sample preparation 38 Place MicroSpin columns in a new set of 2 ml centrifuge tubes 39 Add 200 µl column of C18 Elution Buffer centrifuge at 100 x g for 1 min keep the flow through in the tubes 40 Repeat step 39 once again collecting all eluates in the same tubes Optional if necessary store the tubes wit...

Страница 13: ...44 52 Add 3 µl of Reference Peptide Mix to the Calibration Sample 53 Inject 3 µl of the Calibration Sample for LC MS measurements Please consider the recommended LC settings below depending on your acquisition method Recommended nano flow LC settings for LC MRM Time min Solvent A 1 AcN 0 1 FA H2O Solvent B 3 H2O 0 1 FA AcN 0 100 0 30 65 35 32 0 100 40 0 100 Flow rate 0 3 µl min Sample loading 3µl ...

Страница 14: ...nge to Prepare perspective by clicking on the corresponding icon on the top to recalibrate the PlasmaDive Mini panel for the current LC setup 65 Select the panel PlasmaDive 100 2 1 Mini in the 1 Choose Panel section 66 Select the calibration run used for calibration in step 63 in the 2 Choose LC calibration section The most recently loaded run is shown on top of the table however you may select fr...

Страница 15: ...ur standard LC setup 77 Transfer the calibration run file RAW or WIFF to the computer running SpectroDive 78 Open the Review perspective in SpectroDive by clicking on the corresponding icon on the top 79 To load the calibration run file click on Load raw data and select the run file click Start 80 Verify the calibration linearity and the intensities of iRT peptides in the calibration run Scheduled...

Страница 16: ...t in the field Name 90 Load run files RAW or WIFF and click Start 91 An experiment tab will open In the left panel the LC MS runs are shown as nodes in a tree structure In the status bar you can see the progress of loading the mass spectrometer raw data If you click on the LC MS run node on the left you will see two charts on the right On top in the LC Gradient plot you can see the retention times...

Страница 17: ...on The status of the peak will change to User Manually Integrated 96 3 To return to the SpectroDive initial integration right click on the respective peptide node and select Remove User Peak 96 4 You may make a comment to each peptide by right clicking on the peptide node and selecting Comment C Note 16 97 Peptide node includes the transition group node Transition group nodes are used to group tra...

Страница 18: ...n Comment Comment made while reviewing the run in step 92 optional Protein Id The UniProt accession number of the protein Protein Name The UniProt identifier of the protein Is Identified TRUE or FALSE states whether the protein has been identified with confidence Qvalue 0 01 in the respective sample Injected Amount fmol Absolute amount of the endogenous peptide injected on the column calculated ba...

Страница 19: ...emoved from the samples during the centrifugation step 44 Transfer final samples to single vials and repeat a cold centrifugation 4 C as described in step 44 The first iRT peptide behaves irregularly LC gradient starts with 1 AcN Ensure you use the LC settings as recommended in Section E Because of the high peptide hydrophilicity slight irregularities of the first iRT peptide behaviour still can b...

Страница 20: ...roDive cannot load runs from my Thermo Scientific instrument The Thermo API might not be properly registered Contact Thermo Scientific to download the newest version of the msFileReader API Run the installer in administrator mode An error iRT calibration failed appears when loading my runs into SpectroDive The Reference Peptide Mix was not spiked into samples before LC MS analysis Check the raw fi...

Страница 21: ...amples with the corrected MS method Exported report is empty or does not correspond to the description in the manual A correct SpectroDive Panel Plug in is missing Install a correct Panel Plug in for the kit you are using The incorrect report schema was chosen in step 101 when generating the report Choose the correct report schema and export the report again Some of the protein quantities in my re...

Страница 22: ...rants no license under any of its intellectual property to use or incorporate the enclosed components of this panel with any components not included within this panel except as described in the PlasmaDive Mini Assay Panel Manual and additional protocols available at www biognosys com shop plasmadive mini 2 Other than expressly stated licenses Biognosys makes no warranty that this panel and or its ...

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