Biognosys PlasmaDive Mini User Manual

Download

Second Edition, Version 2.03

April 2017

PlasmaDive™ Mini

Human Plasma Assay Panel for MRM or PRM analysis

MANUAL

Second Edition, Version 2.03

April 2017

PlasmaDive™ Mini Manual, © Biognosys AG, Switzerland

Page 2 of 22

Table of Contents

PlasmaDive™ Mini Assay Panel Components ........................................................... 3

Storage and Quality Control of PlasmaDive™ Mini Kit ............................................... 3

Use Limitations ........................................................................................................... 3

Product Warranty and Satisfaction Guarantee ........................................................... 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 Description ..................................................................................... 18

Troubleshooting Guide ............................................................................................. 19

Trademarks & Limited License Agreement ............................................................... 22

Second Edition, Version 2.03

April 2017

PlasmaDive™ Mini Manual, © Biognosys AG, Switzerland

Page 3 of 22

PlasmaDive™ Mini Assay Panel Components

PlasmaDive™ Mini Kit

Part No: Ki-3012 Sufficient for analysis of 20 samples

Reference Peptide Mix

1x 1.1 ml tube, black cap

Alkylation Solution

1x 10 ml tube, yellow cap

Reduction Stock Solution

1x 0.5 ml tube, orange cap LC Solution

1x 10 ml tube, clear cap

10x Dilution Buffer

1x 10 ml tube, green cap

Denature Buffer

1x 10 ml tube, violet cap

Dissolution Buffer

1x 2.0 ml tube, light blue cap

MicroSpin™ C18 columns

20x C18 columns with the adapter collars PlasmaDive™ Mini MRM and

PRM Panel Plug-ins

Available 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 accordance with Biognosys’ Quality Management System, each lot of the kit is tested against predetermined specifications to ensure consistent product quality.

Use Limitations

PlasmaDive™ Mini Assay Panel is intended for mass spectrometry proteomics applications and research use only. This product is not intended for the diagnostic, prevention, or treatment of a disease. All due care and attention should be exercised in the handling of the products.

Second Edition, Version 2.03

April 2017

PlasmaDive™ Mini Manual, © Biognosys AG, Switzerland

Page 4 of 22

Product Warranty and Satisfaction Guarantee

Biognosys guarantees the performance of the product when following the instructions and protocols described in this product manual. However, the user must determine the suitability of the product for its particular use. Should the product fail to perform satisfactorily due to any reason other than misuse, Biognosys will replace it free of charge. Biognosys reserves the right to change, alter, or modify any product to enhance its performance and design.

If you have questions about product specifications or performance, please contact us at support@biognosys.com. We also encourage you to contact us if you have any suggestions for improving product performance or for its use in new applications and techniques.

Technical Assistance

Our Technical Department is composed of experienced scientists 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 coat, disposable gloves, and protective goggles. For more information, please consult the material safety data sheet (MSDS) available online in convenient and compact PDF format at

www.biognosys.com/shop/plasmadive-mini.

The following risk and safety phrases apply to components of PlasmaDive™ Mini Kit. 10X Dilution Buffer: Harmful if swallowed. Alkylation Solution: Toxic if swallowed, may cause allergy or asthma symptoms or

breathing difficulties if inhaled, may cause an allergic skin reaction, may cause long lasting harmful effects to aquatic life.

Dissolution Buffer: Highly flammable liquid and vapour, causes serious eye irritation. Reduction Stock Solution: Harmful if swallowed, causes skin and eye irritation, may

cause respiratory irritation.

Second Edition, Version 2.03

April 2017

PlasmaDive™ Mini Manual, © Biognosys AG, Switzerland

Page 5 of 22

Introduction: PlasmaDive™ Mini Assay Panel at a Glance

Blood is the most frequently used biological sample in clinical research and routine laboratory diagnostics. Levels of blood proteins reflect the health status of single organs and the body as a whole. Changes in composition of proteins in the blood can be correlated to disease onset or therapy response. Often the relative changes in protein concentrations to each other are the key indicator of a certain condition. Only monitoring the levels of major blood proteins simultaneously makes possible to recognize the “Big Picture” and explain pathological processes going on in the body.

The PlasmaDive™ Mini Assay Panel was designed for targeted proteomics approaches - Multiple Reaction Monitoring (MRM, also called SRM) or Parallel Reaction Monitoring (PRM) - which focus on quantifying predefined sets of proteins with high sensitivity and reproducibility. Biognosys is unique in its ability to perform scheduled highly multiplexed MRM and PRM measurements based on its innovative iRT concept and its specifically developed signal processing software – SpectroDive™. Both, iRT and SpectroDive™, are integral parts of Biognosys’ Assay Panels.

The PlasmaDive™ Mini 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

P02763

A1AG1_HUMAN

Alpha-1-acid glycoprotein 1 (Orosomucoid-1)

P19652

A1AG2_HUMAN

Alpha-1-acid glycoprotein 2 (Orosomucoid-2)

P01009

A1AT_HUMAN

Alpha-1-antitrypsin

P04217

A1BG_HUMAN

Alpha-1B-glycoprotein

P08697

A2AP_HUMAN

Alpha-2-antiplasmin (Serpin F2)

P02750

A2GL_HUMAN

Leucine-rich alpha-2-glycoprotein (LRG)

P01023

A2MG_HUMAN

Alpha-2-macroglobulin (Alpha-2-M)

P01011

AACT_HUMAN

Alpha-1-antichymotrypsin (ACT)

P43652

AFAM_HUMAN

Afamin (Alpha-albumin)

P02768

ALBU_HUMAN

Serum albumin

P35858

ALS_HUMAN

Insulin-like growth factor-binding protein complex acid labile subunit

P02760

AMBP_HUMAN

Protein AMBP

P01019

ANGT_HUMAN

Angiotensinogen (Serpin A8)

P01008

ANT3_HUMAN

Antithrombin-III (Serpin C1)

P02647

APOA1_HUMAN

Apolipoprotein A-I

P02652

APOA2_HUMAN

Apolipoprotein A-II

P06727

APOA4_HUMAN

Apolipoprotein A-IV

P04114

APOB_HUMAN

Apolipoprotein B-100

P02654

APOC1_HUMAN

Apolipoprotein C-I

P02655

APOC2_HUMAN

Apolipoprotein C-II

P02656

APOC3_HUMAN

Apolipoprotein C-III

P05090

APOD_HUMAN

Apolipoprotein D

P02649

APOE_HUMAN

Apolipoprotein E

P02749

APOH_HUMAN

Apolipoprotein H

O14791

APOL1_HUMAN

Apolipoprotein L1

Second Edition, Version 2.03

April 2017

PlasmaDive™ Mini Manual, © Biognosys AG, Switzerland

Page 6 of 22

O95445

APOM_HUMAN

Apolipoprotein M

P43251

BTD_HUMAN

Biotinidase

P02745

C1QA_HUMAN

Complement C1q subcomponent subunit A

P02746

C1QB_HUMAN

Complement C1q subcomponent subunit B

P02747

C1QC_HUMAN

Complement C1q subcomponent subunit C

P00736

C1R_HUMAN

Complement C1r subcomponent

P09871

C1S_HUMAN

Complement C1s subcomponent

P04003

C4BPA_HUMAN

C4b-binding protein alpha chain

P08185

CBG_HUMAN

Corticosteroid-binding globulin (Serpin A6)

O43866

CD5L_HUMAN

CD5 antigen-like (CT-2) (SP-alpha)

P00450

CERU_HUMAN

Ceruloplasmin (Ferroxidase)

P00751

CFAB_HUMAN

Complement factor B

P08603

CFAH_HUMAN

Complement factor H (H factor 1)

P05156

CFAI_HUMAN

Complement factor I

P06276

CHLE_HUMAN

Cholinesterase (EC 3.1.1.8)

P10909

CLUS_HUMAN

Clusterin (Aging-associated gene 4 protein) (Apolipoprotein J)

P06681

CO2_HUMAN

Complement C2

P01024

CO3_HUMAN

Complement C3

P0C0L4

CO4A_HUMAN

Complement C4-A

P01031

CO5_HUMAN

Complement C5

P07357

CO8A_HUMAN

Complement component 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

Fetuin-B

P02671

FIBA_HUMAN

Fibrinogen alpha chain

P02679

FIBG_HUMAN

Fibrinogen gamma chain

P02751

FINC_HUMAN

Fibronectin (FN)

P06396

GELS_HUMAN

Gelsolin (Actin-depolymerizing factor)

P22352

GPX3_HUMAN

Glutathione peroxidase 3

P68871

HBB_HUMAN

Hemoglobin subunit beta

P02042

HBD_HUMAN

Hemoglobin subunit delta (Delta-globin)

P02790

HEMO_HUMAN

Hemopexin (Beta-1B-glycoprotein)

P05546

HEP2_HUMAN

Heparin cofactor 2

P00738

HPT_HUMAN

Haptoglobin

P00739

HPTR_HUMAN

Haptoglobin-related protein

P04196

HRG_HUMAN

Histidine-rich glycoprotein

P05155

IC1_HUMAN

Plasma protease C1 inhibitor

P01876

IGHA1_HUMAN

Ig alpha-1 chain C region

P01877

IGHA2_HUMAN

Ig alpha-2 chain C region

P01857

IGHG1_HUMAN

Ig gamma-1 chain C region

P01859

IGHG2_HUMAN

Ig gamma-2 chain C region

P01860

IGHG3_HUMAN

Ig gamma-3 chain C region (HDC)

P01871

IGHM_HUMAN

Ig mu chain C region

Second Edition, Version 2.03

April 2017

PlasmaDive™ Mini Manual, © Biognosys AG, Switzerland

Page 7 of 22

P05154

IPSP_HUMAN

Plasma serine protease inhibitor

P19827

ITIH1_HUMAN

Inter-alpha-trypsin inhibitor heavy chain H1

P19823

ITIH2_HUMAN

Inter-alpha-trypsin inhibitor heavy chain H2

Q14624

ITIH4_HUMAN

Inter-alpha-trypsin inhibitor heavy chain H4

P29622

KAIN_HUMAN

Kallistatin (Kallikrein inhibitor)

P03952

KLKB1_HUMAN

Plasma kallikrein

P01042

KNG1_HUMAN

Kininogen-1

P36955

PEDF_HUMAN

Pigment epithelium-derived factor (PEDF)

Q96PD5

PGRP2_HUMAN

N-acetylmuramoyl-L-alanine amidase

P02776

PLF4_HUMAN

Platelet factor 4 (Oncostatin-A)

P00747

PLMN_HUMAN

Plasminogen

P27169

PON1_HUMAN

Serum paraoxonase

Q92954

PRG4_HUMAN

Proteoglycan 4

P02753

RET4_HUMAN

Retinol-binding protein 4

P35542

SAA4_HUMAN

Serum amyloid A-4 protein

P49908

SEPP1_HUMAN

Selenoprotein 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 Notes before Start

Before starting with the sample preparation, read through the steps carefully and make sure all the required reagents and equipment are available.

Use minimally LC-grade solvents and water throughout the protocol to prepare buffers and solutions.

The kit components are sufficient for 20 human plasma samples.

Sample Requirements

Human plasma or serum, 10 µl per sample. Reserve one suitable sample for calibration run. If it is not possible to dedicate one

sample for a calibration run, a separate LC-MS sample for calibration can be prepared following instructions in Section E of Sample Preparation Procedure & LC-MRM/PRM Analysis.

Second Edition, Version 2.03

April 2017

PlasmaDive™ Mini Manual, © Biognosys AG, Switzerland

Page 8 of 22

SpectroDive™ Software and PlasmaDive™ Mini Panel Plug-in

An integral part of the PlasmaDive™ Assay Panel is the SpectroDive™ software that enables easy MS method setup and automated signal processing of your LCMRM/PRM measurements. SpectroDive™ supports all Biognosys’ Assay Panels. For each panel corresponding software plug-ins are required.

SpectroDive™ is free for academic researchers and can be obtained at

www.biognosys.com/shop/spectrodive. The downloading instructions and your

personal License Key will be sent to your e-mail account within one working day after the registration. Enterprise licenses are available for industry customers at

www.biognosys.com/shop/spectrodive. PlasmaDive™ Mini Panel MRM and PRM

plug-ins will be provided to you by the Biognosys team upon request to

support@biognosys.com.

SpectroDive™ can be installed and run on a notebook or desktop computer. The minimal system requirements are: Windows 7 or 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 installation

SpectroDive™ Activation When running SpectroDive™ for the first time you will be prompted to activate the

software. For activation use your personal License Key from the e-mail with the downloading instructions.

An internet connection is required for the on-line SpectroDive™ activation. If your computer is not connected to the internet or the activation is blocked by your firewall, please follow the SpectroDive™ instructions for the off-line activation.

PlasmaDive™ Mini Panel Plug-in Import (MRM or PRM)  In SpectroDive™ select the ‘Prepare’ perspective by clicking on the corresponding

icon on the top.

 Import the .kit file for PlasmaDive™ Mini by clicking on ‘Import New Panel’ in the low

part of ‘1) Choose Panel’ section on the left.

 The panel “PlasmaDive™ 100.2.1 Mini” or “PlasmaDive™ 100.2.1 Mini PRM” is now

available in the ‘Panel’ folder.

Second Edition, Version 2.03

April 2017

PlasmaDive™ Mini Manual, © Biognosys AG, Switzerland

Page 9 of 22

Additionally Required Laboratory Equipment and Consumables

Single channel pipettes (0.5 µl – 1000 µl) with corresponding tips

1.5 ml and 2 ml plastic tubes

Glass syringe (100 µl)

pH paper (recommended universal pH indicator paper pH 1-10 with colour scale) or pH meter with small combined glass electrode

Vortex mixer

15 ml plastic tubes

Benchtop centrifuge

Thermomixer at +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/v) TFA solution.

10% (v/v) TFA solution, prepare at least 1 ml for 20 samples (Note 1, Note 2)

Methanol, at least 8 ml for 20 samples

Water

Acetonitrile

C18 Cleaning Solution, prepare at least 8 ml for 20 samples (Note 1)

 80% (v/v) Acetonitrile  0.1% (v/v) TFA (Note 2, Note 3)  In water

Second Edition, Version 2.03

April 2017

PlasmaDive™ Mini Manual, © Biognosys AG, Switzerland

Page 10 of 22

C18 Washing Solution, prepare at least 30 ml for 20 samples (Note 1)

 1% (v/v) Acetonitrile  0.1% (v/v) TFA (Note 2, Note 3)  In water

C18 Elution Solution, prepare at least 10 ml for 20 samples (Note 1)

 50% (v/v) Acetonitrile  0.1% (v/v) TFA (Note 2, Note 3)  In water

Sample Preparation Procedure & LC-MRM/PRM Analysis

A. Denaturation, reduction and alkylation

1. Dissolve 10x Dilution Buffer with water to a total volume of 5 ml (Note 4), vortex until solubilized.

2. Dissolve Reduction Stock Solution with 250 µl water, vortex, briefly spin down.

3. Prepare Denature Buffer:

3.1. Add 500 µl of 10x Dilution Buffer to the Denature Buffer tube. Keep 10x Dilution Buffer in fridge until further usage in step 14.

3.2. Add 25 µl of Reduction Stock Solution to the Denature Buffer tube.

3.3. Fill up Denature Buffer with 2.3 ml of water (Note 4, Note 5).

4. Using a single channel pipette, add 90 µl/tube of Denature Buffer to 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 with water to 3 ml (Note 4), vortex, briefly spin down (Note 7).

10. Using a single channel pipette, add 16 µl/tube of Alkylation Solution.

11. Gently shake the samples on thermomixer for 1 min at room temperature.

12. Briefly spin down to collect all liquid at the bottom of the tubes.

13. Incubate the samples at room temperature in the dark for 30 min.

Note 4

To solubilize the provided solid components, dissolve first in a small amount of the stated solvent and fill-up to final volume carefully.

Note 5

Warm up the tube to help solubilizing the reagent by holding the tube in warm (<40°C) tap water.

Note 6

Thawed plasma samples should not be left without Denature Buffer at room temperature for longer than 5 minutes.

Note 7

Light sensitive, prepare shortly before usage and keep in dark.

Second Edition, Version 2.03

April 2017

PlasmaDive™ Mini Manual, © Biognosys AG, Switzerland

Page 11 of 22

B. Dilution

14. Prepare 1x Dilution Buffer by mixing in a separate 15 ml tube 1 ml of 10x Dilution Buffer and 9 ml of water.

15. Using a single channel pipette, add 150 µl/tube of 1x Dilution Buffer to a new set of 1.5 ml tubes.

16. Using a single channel pipette, add 15 µl/tube of denatured plasma sample (from Section A, step 13).

Optional: you might freeze remaining sample in the tubes for a repeated analysis.

17. Check pH to be 8–9 in a few samples by pipetting 0.5 µl onto a pH paper or by using a pH-electrode (Note 8).

17.1. If pH is below 8, adjust it in all samples using 5 µl of 10x Dilution Buffer.

17.2. Check again in a few wells, repeat steps 17.1 and 17.2 if necessary.

C. Digestion using endoprotease trypsin

18. Thaw trypsin (0.4 µg/µl) and spin down briefly.

19. Add 5 µl/tube of trypsin to the tubes with samples from Section B 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 pipetting 0.5 µl onto a pH paper or by using a pH-electrode.

24.1. If pH is above 2, adjust it in all samples adding 5 µl of 10% (v/v) TFA solution.

24.2. Check again in a few samples, repeat steps 24.1 and 24.2 if necessary.

25. Gently shake the samples on thermomixer for 1 min at room temperature.

26. Briefly spin down to collect all liquid at the bottom of the tubes.

Optional: if convenient store samples at -20°C until C18 clean-up.

D. Sample clean-up using MicroSpin columns

Spin columns preparation

27. Place 20 MicroSpin columns in a new set of 2 ml centrifuge tubes.

Note 8

Avoid sample crosscontamination by using fresh tips or cleaning the electrode with water for every sample.

Note 9

Foaming is possible; add 10% (v/v) TFA solution slowly.

Second Edition, Version 2.03

April 2017

PlasmaDive™ Mini Manual, © Biognosys AG, Switzerland

Page 12 of 22

28. Add 200 µl of methanol into each column, centrifuge it at 100 x g for 1 min and discard flow-through.

29. Add 200 µl/column of C18 Cleaning Solution, centrifuge at 100 x g for 1 min, and discard flow-through.

30. Add 200 µl/column of C18 Washing Solution, centrifuge at 100 x g for 1 min, and discard flow-through.

31. Repeat step 30 two more times. Make sure the there is no washing solution left on the column material surface after the last wash.

Sample loading to spin columns

32. Centrifuge (thawed) samples from Section C at 16000 x g for 1 min.

33. Place MicroSpin columns in a new set of 2 ml centrifuge tubes.

34. Load samples (supernatant) into the MicroSpin columns, centrifuge at 100 x g for 1 min, do not discard flow-through (Note 10).

35. Repeat step 34 by re-loading the flow-through collected in step

34. Discard the flow through (Note 10).

36. Add 200 µl/column of C18 Washing Solution, centrifuge at 100 x 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 with samples at -20°C until drying.

41. Dry down the combined eluates using a vacuum centrifuge (Note 11).

E. LC-MS sample preparation & LC settings

42. Dissolve dried samples (step 41) in 225 µl/tube of LC Solution by pipetting up and down.

43. Vortex the tubes thoroughly or shake them on thermomixer for 10 min at room temperature.

44. Centrifuge the dissolved samples at 4°C and 20`000 x g for 20 min.

45. Transfer 6 µl of sample supernatants to LC-MS vials. Store remaining samples at -20°C.

46. Prepare Reference Peptide Mix by adding to the glass vial 10 µl of Dissolution Buffer; vortex briefly.

Note 10

You may increase the centrifugation force up to 400 x g in case of low flow rates through the MicroSpin columns. The force can be increased to max 1’000 x g.

Note 11

Dried samples can be stored at -20°C until usage.

Second Edition, Version 2.03

April 2017

Page 13 of 22

47. Add 50 µl of LC Solution to the Reference Peptide Mix.

48. Vortex the Reference Peptide Mix, sonicate for 5 min if possible.

49. Add 2 µl/sample of Reference Peptide Mix to each LC-MS vial using a single channel pipette.

50. Inject 3 µl/sample for all LC-MS measurements. Please consider the recommended LC settings below.

If you have included a Calibration Sample in your sample set continue with Section F. Otherwise follow the steps 51-53.

51. Prepare a Calibration Sample by pooling into a separate LC-

MS vial of 1 µl from 9 randomly selected samples from step 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

100%

65%

35%

100%

Flow rate

0.3 µl/min

Sample loading

3µl/injection (approx. 1µg protein/injection)

Recommended nano-flow LC settings for LC-PRM:

Time, min

Solvent A 1% AcN / 0.1% FA / H2O

Solvent B 3% H2O / 0.1% FA / AcN

100%

120

65%

35%

122

100%

130

100%

Flow rate

0.3 µl/min

Sample loading

3µl/injection (approx. 1µg protein/injection)

F. LC-MS retention time (RT) calibration and scheduled

measurement of PlasmaDive™ Mini Assay Panel

If you are acquiring your samples in PRM mode, continue at step 71.

LC-MRM retention time calibration

Second Edition, Version 2.03

April 2017

Page 14 of 22

54. Open the “Prepare” perspective in SpectroDive™ by clicking on the corresponding icon top left.

55. In section “1) Choose panel” select iRT-Kit from the folder “LC calibration”.

56. In section “3) Export Method” select your instrument and software version, set Dwell Time to 10-20 ms.

57. Export the method (transition file) as comma-separated file by

clicking on ‘Export Method …’ and save it to the computer

connected to your LC-MS system.

58. Import the method (transition file) to your unscheduled LCMRM (also called SRM) instrument method.

59. Measure your calibration sample in unscheduled MRM mode on your standard LC setup.

60. Transfer the calibration run file (.RAW or .WIFF) to the computer running SpectroDive™.

61. Open the ‘Review’ perspective in SpectroDive™ by clicking on the corresponding icon on the top.

62. To load the calibration run file click on ‘Load raw data…’ and select the run file, click ‘Start’.

63. Verify the calibration linearity and the intensities of iRT peptides in the calibration run.

Scheduled measurement of PlasmaDive™ Mini Assay Panel

64. Change 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 from other previously loaded runs.

67. Adjust the instrument and method settings in ‘3) Export Method’ section (Note 12).

67.1. Change to a scheduled method on the pull-down menu ‘Scheduling’.

67.2. Adjust the retention time window in minutes in the field

‘Window (min)’ in order to not to exceed a maximum

number of concurrent transitions (e.g. assuming a constant cycle time of 2.5 s, one should not exceed maximal 250 concurrent transitions since this may lead to a dwell time of lower than 10 ms).

68. Export the method (transition file) by clicking on ‘Export Method …’ and save it to a location of your choice.

69. Attach the transition file to the schedule instrument method of choice and start the data acquisition of your samples from step 50.

Note 12

The plot in section ‘3) Export Method’ shows the number of concurrent transitions or precursors (i.e. the number of transitions or precursors the instrument has to cycle through at a certain time in the gradient).

Second Edition, Version 2.03

April 2017

Page 15 of 22

69.1. Make sure that you use the exact same LC setup and gradient as used for the calibration.

69.2. Make sure that you use constant cycle time.

70. Measure all samples in your experiment using the same scheduled instrument method (Note 13).

70.1. Continue with data analysis at step 88

LC-PRM retention time calibration

71. Open the “Prepare” perspective in SpectroDive™ by clicking

on the corresponding icon top left.

72. In section “1) Choose panel” select “PRM iRT-Kit” from the folder “LC calibration”.

73. In section “3) Export Method” select your instrument and software version

74. Export the method (inclusion list) as comma-separated file by

clicking on ‘Export Method …’ and save it to the computer

connected to your LC-MS system.

75. Import the inclusion list to your unscheduled PRM instrument method.

76. Measure your calibration sample in unscheduled PRM mode on your 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 measurement of PlasmaDive™ Mini Assay Panel

81. Change to ‘Prepare’ perspective by clicking on the corresponding icon on the top to recalibrate the PlasmaDive™ Mini panel for the current LC setup.

82. Select the panel ‘PlasmaDive™ 100.2.1 Mini.PRM’ in the ‘1) Choose Panel’ section

83. 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 from other previously loaded runs.

84. Adjust the instrument and method settings in ‘3) Export Method’ section (Note 12).

Note 13

In case major system changes (e.g. new column) a new calibration run has to be performed and a new scheduled instrument method has to be generated according to the Section F.

Second Edition, Version 2.03

April 2017

Page 16 of 22

84.1. Change to a scheduled method on the pull-down menu ‘Scheduling’.

84.2. Adjust the retention time window in minutes in the field

‘Window (min)’ in order to not to exceed a maximum

number of 80 concurrent precursors

85. Export the method (inclusion list) by clicking on ‘Export Method …’ and save it to a location of your choice.

86. Import the inclusion list into the PRM instrument method of choice (Note 14) and start the data acquisition of your samples from step 50.

86.1. Make sure that you use the exact same LC setup and gradient as used for the calibration.

87. Measure all samples in your experiment using the same scheduled instrument method (Note 13).

G. PlasmaDive™ Mini Assay Panel data analysis

88. After the panel measurements are done, transfer the raw files

to the computer running SpectroDive™.

89. Change to the ‘Review’ perspective and click on ‘Load Raw

Data…’ directly or first on the ‘plus’ sign on top to generate a

new experiment tab.

Optional: define a name for the experiment 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 of the iRT peptides indicating the linearity of your LC-gradient. In the lower plot you can see the summed up ion traces for all the peptides that were measured.

92. You may make a comment to each LC-MS run e.g. assigning a

sample ID or general remarks by right clicking on the LC-MS run name in the left panel and selecting ‘Comment (C)’.

93. By clicking on the LC-MS run node, the panels measured in

this run will appear.

94. By clicking on a panel node the peptide nodes will appear.

95. When you click on a peptide node two graphical panels on the

right will appear.

95.1. The top right panel displays all the transitions of this peptide as well as the peak boundaries and the Qvalue.

Note 14

We recommend to use a resolution of 17.5k (Orbitrap) or 30k (Orbitrap HF) which corresponds to a max fill time of 55ms per precursor, due to the high level of multiplexing of the assay panel.

Second Edition, Version 2.03

April 2017

Page 17 of 22

The Qvalue indicates the confidence of the endogenous (light) peptide identification (lower Qvalue indicates higher confidence) (Note 15).

95.2. The bottom right panel shows the sum of all transitions together with the peak boundaries as well as quantitative information about this peptide.

95.3. You can zoom in the plots and perform various actions by right clicking on the plot. Right clicking also enables you to set the scale back to default after zooming.

95.4. You can select from various plots to be displayed in the pull-down menus on top for ‘Upper panel’ and ‘Lower

panel’ e.g. to ‘Alignment view’ in order to review one

peptide across all runs.

96. Review the peptide signals (User Verification).

96.1. When a peptide is selected, you can manually accept or reject it. Use the menu that appears by right clicking on the peptide node or toggle using the keyboard hotkeys “A” and “R”. If no action is performed, the peptides remain in the analysis as unchecked (no user verification).

96.2. To manually change peak integration, readjust the green integration boundaries (green box). Drag and drop the border to the correct location. 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 transitions according to isotopic labelling. If you select the transition group node on the left you will again see two graphical panels on the right. The top panel shows the transitions of this transition group in different colours. The bottom panel displays information about the peak. You can see quantitative as well as chromatographic information.

98. One level below you can find the transition node. If transition node is selected you will see again two graphical panels on the left that show the information specific to this transition.

99. You can use multiple filtering options at the bottom of the left panel to select specific nodes of the tree (e.g. in Qvalue filter

when ‘Value’ is set ≤ 0.01, only the peptides with Qvalues ≤0.01 are displayed).

Generation of report

100. Switch to the ‘Report’ perspective.

101. Choose ‘PlasmaDive 100.2’ schema on the left.

Note 15

SpectroDive™ by

default qualifies a protein as identified when Qvalue is ≤0.01.

Note 16

This is not equal to the comments that can be made to each run.

Second Edition, Version 2.03

April 2017

Page 18 of 22

102. Export the report to a tab separated table file (.xls file). This

file format can be further edited with e.g. Microsoft Excel.

Report Columns Description

The SpectroDive™ report is run based. Each row in the report corresponds to a run. The following columns are included into the report:

Column name

Description

Experiment Name

The name of experiment that have been specified while loading the data. If the user hasn’t specified anything, SpectroDive™ automatically assigns a default name.

Run Name

The name(s) of run file(s) without the .RAW or .WIFF extension.

Run 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 based on the following assumption:

 Section E of the protocol above was exactly followed.

Plasma Concentration [µg/µl]

Concentration of the protein in the original 10 µl plasma sample calculated based on the following assumptions:

 No protein / peptide losses during the sample preparation;  Peptide amount is representative for the corresponding

protein;

 Protein molecular weight is based on amino acid

sequence;

 Steps 1-102 of the protocol above were exactly followed.

Second Edition, Version 2.03

April 2017

Page 19 of 22

Qvalue

Qvalue assigned by SpectroDive™ (see step 95.1).

User Verified

Specifies whether the user has accepted, rejected or not checked the protein manually in the respective sample (see step 96.1).

User Integrated

TRUE or FALSE, states whether the peptide peak has been manually integrated by the user.

Protein Comment

Comment made while reviewing the peptide in step 96.4.

Troubleshooting Guide

This troubleshooting guide may be helpful in solving issues that may arise.

Issue

Possible Cause

Biognosys PlasmaDive Mini User Manual

Specifications and Main Features

Frequently Asked Questions

User Manual