TITLE: Protocol for Data Independent Acquisition - Mass spectrometry analysis – a DIA-based Organelle Proteomics
AUTHORS: Raja Sekhar Nirujogi, Rotimi Fasimoye, Toan K Phung, Dario R Alessi

[DESCRIPTION]
Purification of intact organelles by previously described methods (dx.doi.org/10.17504/protocols.io.bybjpskn; dx.doi.org/10.17504/protocols.io.6qpvrdjrogmk/v1) allows to profile the organelle proteome using quantitative mass spectrometry. Here we provide a detailed protocol for the Data Independent Acquisition (DIA)-based mass spectrometry (MS) data acquisition method for proteomic profiling of the Golgi. This includes a description of how to construct the nano Liquid chromatography and DIA MS methods as well as a Data Dependent Acquisition (DDA) strategy to generate deep spectral libraries to be able to use in searching the DIA data. In addition, we provide detailed search parameters for database search for both DDA and DIA and downstream MS data analysis.

[STEPS]
SECTION: High-pH Reversed-phase Liquid Chromatography fractionation of pooled Golgi-tag IP peptides to generate Spectral library:
1. Take ~5 µg of peptide digest from each of the Golgi-tag IP and Control-IP sample.
SECTION: High-pH Reversed-phase Liquid Chromatography fractionation of pooled Golgi-tag IP peptides to generate Spectral library:
2. Vacuum dry the pooled samples.
SECTION: High-pH Reversed-phase Liquid Chromatography fractionation of pooled Golgi-tag IP peptides to generate Spectral library:
3. Dissolve the peptide digest by adding 120 µL of High-pH Solvent-A (10 millimolar (mM) Ammonium formate pH 10.0). Place the sample on a Thermomixer with an agitation at 1800 rpm for 30 min.
SECTION: High-pH Reversed-phase Liquid Chromatography fractionation of pooled Golgi-tag IP peptides to generate Spectral library:
4. Centrifuge the sample at high speed (17000 x g) for 5 min at Room temperature.
SECTION: High-pH Reversed-phase Liquid Chromatography fractionation of pooled Golgi-tag IP peptides to generate Spectral library:
5. Take 0.5 µL of the sample and verify the pH and transfer the sample into LC-vial.
SECTION: High-pH Reversed-phase Liquid Chromatography fractionation of pooled Golgi-tag IP peptides to generate Spectral library:
6. Ensure the LC-solvent are as Solvent-A (10 millimolar (mM) Ammonium formate pH 10.0); Solvent-B (90% ACN (v/v) in 10 millimolar (mM) Ammonium formate pH 10.0).
 

SECTION: High-pH Reversed-phase Liquid Chromatography fractionation of pooled Golgi-tag IP peptides to generate Spectral library:
7. Prepare the LC method by following the below gradient:
 
     Time (minutes)      Nano pump Flow rate (µl/min)      % Of Solvent-B        0.0      0.100      3.0        5.0      0.100      7.0        5.5      0.100      7.0        10.0      0.100      10.0        50.0      0.100      40.0        55.0      0.100      90.0        62.0      0.100      90.0        62.5      0.100      3.0        70.0      0.100      3.0        70.1      0.0100      3.0   
 
SECTION: High-pH Reversed-phase Liquid Chromatography fractionation of pooled Golgi-tag IP peptides to generate Spectral library:
8. Set the fraction collection time as Start time 305 s and End time 62 min.
SECTION: High-pH Reversed-phase Liquid Chromatography fractionation of pooled Golgi-tag IP peptides to generate Spectral library:
9. Collect a total of 45 fractions by keeping the fraction collection for 75 s for each fraction.
SECTION: High-pH Reversed-phase Liquid Chromatography fractionation of pooled Golgi-tag IP peptides to generate Spectral library:
10. Transfer the fractions into a pre-labelled 1.5 mL protein lo binding tubes.
SECTION: High-pH Reversed-phase Liquid Chromatography fractionation of pooled Golgi-tag IP peptides to generate Spectral library:
11. Vacuum dry the samples and freeze in -20 freezer until the LC-MS/MS analysis.
SECTION: Single shot DIA acquisition on Orbitrap Exploris 480:
12. Dissolve vacuum dried peptides in 60 µL of LC buffer (3% ACN in 0.1% Formic acid) and place the samples on a Thermomixer and mix them at 1800 rpm at Room temperature for about 30 min.
SECTION: Single shot DIA acquisition on Orbitrap Exploris 480:
13. Take 4 µg equivalent of peptide digest and spike 1 µL of iRT peptide mix. Adjust the total volume of the sample anywhere between 5 µL to 15 µL but don’t exceed 15 µL. Transfer the sample into glass insert and place them on LC vial.
 

SECTION: Single shot DIA acquisition on Orbitrap Exploris 480:
14. Construct LC and vDIA MS method as described below using Xaclibur software integrated in Thermo Orbitrap Exploris 480 MS acquisition software suite.
SECTION: Single shot DIA acquisition on Orbitrap Exploris 480:
15. Ensure   trap column (C18, 5 μm, 100A°, 100 µ,   Nano-viper column # 164564, Thermo Scientific) and   analytical column (C18, 5 micromolar (µM),  , 100Aº Easy nano spray column # ES903, Thermo Scientific) are equilibrated and verify the column performance by injecting 50 ng HeLa or another standard digest.

SECTION: Single shot DIA acquisition on Orbitrap Exploris 480:
16. Nano LC gradient for 145 min DIA analysis:
 
     Time (minutes)      Nano pump Flow rate (µl/min)      % Of Solvent-B        0.0      0.250      3.0        12.0      0.250      7.0        115.0      0.250      25.0        129.0      0.250      37.0        130.0      0.250      95.0        135.30      0.250      95.0        135.80      0.250      3.0        145.0      0.250      3.0        145.0      Stop Run   
 
SECTION: Single shot DIA acquisition on Orbitrap Exploris 480:
17. Mass spectrometer parameters: Refer below settings to construct variable DIA method:
  
            Method duration  145 min          MS Global settings:                 Infusion mode:   Liquid Chromatography          Expected LC peak width (s):  20          Advanced Peak determination:  TRUE          Default charge state:  3          Internal mass calibration:  off  Note: If needed   enable user defined calibrant ion (Polysilaxolane 445.120025 or enable   Easy-IC option      Full scan settings:                Orbitrap resolution:  120000          Scan range (m/z):  375-1500          RF lens (%):  40          AGC target:  Custom     
 
     Scheme of vDIA windows mass list table:                                     m/z      z      Isolation Window (mz)               383.375      3      66.8               423      3      13.5               435      3      11.5               446.5      3      12.5               458      3      11.5               469      3      11.5               480      3      11.5               490.5      3      10.5               501      3      11.5               512      3      11.5               523      3      11.5               533.5      3      10.5               544      3      11.5               554.5      3      10.5               565      3      11.5               575.5      3      10.5               586      3      11.5               597.5      3      12.5               609.5      3      12.5               621.5      3      12.5               633      3      11.5               645      3      13.5               657.5      3      12.5               670.5      3      14.5               684      3      13.5               697      3      13.5               710.5      3      14.5               725.5      3      16.5               741      3      15.5               756.5      3      16.5               773.5      3      18.5               791      3      17.5               808.5      3      18.5               827      3      19.5               846.5      3      20.5               866.5      3      20.5               887.5      3      22.5               910.5      3      24.5               935.5      3      26.5               962.5      3      28.5               992      3      31.5               1025      3      35.5               1063      3      41.5               1108.5      3      50.5               1391.625      3      516.8   

SECTION: Single shot DIA acquisition on Orbitrap Exploris 480:
18. Export the MS raw data for database searches by library-free (direct DIA) or library-based as illustrated in the workflow with Biognosys Spectronaut software suite.
 

SECTION: Data Dependent Acquisition (DDA) MS analysis to generate Spectral library:
19. Dissolve vacuum dried peptides of each fraction in 60 µL of LC buffer (3% ACN in 0.1% Formic acid) and place the samples on a Thermomixer and mix them at 1800 rpm at Room temperature for about 30 min.
SECTION: Data Dependent Acquisition (DDA) MS analysis to generate Spectral library:
20. Take 1 µg equivalent of peptide digest and Spike 1 µL of iRT peptide mix. Adjust the total volume of the sample anywhere between 5 µL to 15 µL but don’t exceed 15 µL. Transfer the sample into glass insert and place them on LC vial.
 

SECTION: Data Dependent Acquisition (DDA) MS analysis to generate Spectral library:
21. Ensure   trap column (C18, 5 μm, 100Ao, 100 µ,   Nano-viper column # 164564, Thermo Scientific) and   analytical column (C18, 5 micromolar (µM),  , 100Ao Easy nano spray column # ES903, Thermo Scientific) are equilibrated and verify the column performance by injecting 50 ng HeLa or another standard digest.

SECTION: Data Dependent Acquisition (DDA) MS analysis to generate Spectral library:
22. Nano LC gradient for 85 min DDA analysis:
     Time (minutes)      Nano pump Flow rate (µl/min)      % Of Solvent-B        0.0      0.300      3.0        7.0      0.300      7.0        60.0      0.300      22.0        70.0      0.300      35.0        71.0      0.300      95.0        78.0      0.300      95.0        79.0      0.300      3.0        85.0      0.300      3.0        85.0      Stop Run   

SECTION: Data Dependent Acquisition (DDA) MS analysis to generate Spectral library:
23. Mass spectrometer parameters: Refer below settings to construct DDA method:
 
     Method duration      85 min               MS Global settings:                              Infusion mode:       Liquid Chromatography               Expected LC peak width (s):      15               Advanced Peak determination:      TRUE               Default charge state:      2               Internal mass calibration:      off          Full scan settings:                             Orbitrap resolution:      60000               Scan range (m/z):      350-1200               RF lens (%):      40               AGC target:      Custom               Normalized AGC target (%):      300               Maximum injection Time mode:      Custom               Maximum injection Time (ms):      28               Micorscans:      1               Data type:      Profile               Polarity:      Positive        Filters:                      MIPS      Monoisotopic peak determination:      Peptide               Relax restrictions when too few precursors are found:      FALSE        Intensity      Filter Type:         Intensity Threshold               Intensity Threshold:      1.00E+04        Charge State      Include charge state(s):         2 to 6               Include undetermined charge states:           False        Dynamic Exclusion      Dynamic Exclusion Mode:      Custom               Exclude after n times:      1               Exclusion duration (s):       45               Mass Tolerance:      ppm               Low:      10               High      10               Exclude isotopes:         TRUE               Perform dependent scan on single charge state per precursor only:         FALSE        Data Dependent      Data Dependent Mode:      Cycle Time               Time between Master Scans (sec):      3        ddMS2 settings      Isolation Window (m/z):      1.2               Isolation Offset:      Off               Collision Energy Mode:      Fixed               Collision Energy Type:      Normalized               HCD Collision Energy (%):      30               Orbitrap resolution:      15000               Scan range mode:      Auto               Scan Range (m/z):      200 - 1200               AGC target:      Custom               Normalized AGC target (%):      100               Maximum injection Time mode:      Custom               Maximum injection Time (ms):      85               Micorscans:      1               Data type:      Centroid               Polarity:      Positive   
 
SECTION: Database searches with MaxQuant for Data Dependent Acquisition (DDA) MS analysis to generate Spectral library:
24. Export Raw MS data to a Windows server to perform database searches using MaxQuant. Refer the below search parameters for the search.
 
 
     5.1)   Parameter      Value        Version      1.6.10.0        Include contaminants      TRUE        PSM FDR      0.01        PSM FDR Crosslink      0.01        Protein FDR      0.01        Site FDR      0.01        Use Normalized Ratios for Occupancy      TRUE        Min. peptide Length      7        Min. score for unmodified peptides      0        Min. score for modified peptides      40        Min. delta score for unmodified peptides      0        Min. delta score for modified peptides      6        Min. unique peptides      0        Min. razor peptides      1        Min. peptides      1        Use only unmodified peptides and      TRUE        Modifications included in protein quantification      Oxidation   (M);Acetyl (Protein N-term)        Peptides used for protein quantification      Razor        Discard unmodified counterpart peptides      TRUE        Label min. ratio count      2        Use delta score      FALSE        iBAQ      TRUE        iBAQ log fit      TRUE        Match between runs      TRUE        Matching time window [min]      0.7        Match ion mobility window [indices]      0.05        Alignment time window [min]      20        Alignment ion mobility window [indices]      1        Find dependent peptides      FALSE        Fasta file      D:\Database\20200723-Human-Uniprot.fasta        Decoy mode      revert        Include contaminants      TRUE        Advanced ratios      TRUE        Fixed andromeda index folder               Temporary folder               Combined folder location               Second peptides      TRUE        Stabilize large LFQ ratios      FALSE        Separate LFQ in parameter groups      FALSE        Require MS/MS for LFQ comparisons      FALSE        Calculate peak properties      FALSE        Main search max. combinations      200        Advanced site intensities      TRUE        Write msScans table      TRUE        Write msmsScans table      TRUE        Write ms3Scans table      FALSE        Write allPeptides table      TRUE        Write mzRange table      TRUE        Write pasefMsmsScans table      FALSE        Write accumulatedPasefMsmsScans table      FALSE        Max. peptide mass [Da]      4600        Min. peptide length for unspecific search      8        Max. peptide length for unspecific search      25        Razor protein FDR      TRUE        Disable MD5      FALSE        Max mods in site table      3        Match unidentified features      FALSE        Epsilon score for mutations               Evaluate variant peptides separately      TRUE        Variation mode      None        MS/MS tol. (FTMS)      20 ppm        Top MS/MS peaks per Da interval. (FTMS)      12        Da interval. (FTMS)      100        MS/MS deisotoping (FTMS)      TRUE        MS/MS deisotoping tolerance (FTMS)      7        MS/MS deisotoping tolerance unit (FTMS)      ppm        MS/MS higher charges (FTMS)      TRUE        MS/MS water loss (FTMS)      TRUE        MS/MS ammonia loss (FTMS)      TRUE        MS/MS dependent losses (FTMS)      TRUE        MS/MS recalibration (FTMS)      TRUE        MS/MS tol. (ITMS)      0.5 Da        Top MS/MS peaks per Da interval. (ITMS)      8        Da interval. (ITMS)      100        MS/MS deisotoping (ITMS)      FALSE        MS/MS deisotoping tolerance (ITMS)      0.15        MS/MS deisotoping tolerance unit (ITMS)      Da        MS/MS higher charges (ITMS)      TRUE        MS/MS water loss (ITMS)      TRUE        MS/MS ammonia loss (ITMS)      TRUE        MS/MS dependent losses (ITMS)      TRUE        MS/MS recalibration (ITMS)      FALSE        MS/MS tol. (TOF)      40 ppm        Top MS/MS peaks per Da interval. (TOF)      10        Da interval. (TOF)      100        MS/MS deisotoping (TOF)      TRUE        MS/MS deisotoping tolerance (TOF)      0.01        MS/MS deisotoping tolerance unit (TOF)      Da        MS/MS higher charges (TOF)      TRUE        MS/MS water loss (TOF)      TRUE        MS/MS ammonia loss (TOF)      TRUE        MS/MS dependent losses (TOF)      TRUE        MS/MS recalibration (TOF)      FALSE        MS/MS tol. (Unknown)      20 ppm        Top MS/MS peaks per Da interval. (Unknown)      12        Da interval. (Unknown)      100        MS/MS deisotoping (Unknown)      TRUE        MS/MS deisotoping tolerance (Unknown)      7        MS/MS deisotoping tolerance unit (Unknown)      ppm        MS/MS higher charges (Unknown)      TRUE        MS/MS water loss (Unknown)      TRUE        MS/MS ammonia loss (Unknown)      TRUE        MS/MS dependent losses (Unknown)      TRUE        MS/MS recalibration (Unknown)      FALSE        Site tables      Deamidation   (NQ)Sites.txt;Oxidation (M)Sites.txt;Phospho (STY)Sites.txt   
 
SECTION: Database searches with Biognosys Spectronaut for Data Dependent Independent Acquisition (DIA) MS analysis (Library free and Library-based search):
25. Import the msms.txt file form the MaxQuant search output files into Spectronaut to generate a Spectral library.
 

SECTION: Database searches with Biognosys Spectronaut for Data Dependent Independent Acquisition (DIA) MS analysis (Library free and Library-based search):
26. Alternatively perform a Pulsar search of DDA data to generate a library.
SECTION: Database searches with Biognosys Spectronaut for Data Dependent Independent Acquisition (DIA) MS analysis (Library free and Library-based search):
27. As illustrated in the workflow we recommend doing a direct-DIA or Library free search using Human Uniprot FAST file to construct a hybrid library. Enable search archive option during the direct-DIA search.
SECTION: Database searches with Biognosys Spectronaut for Data Dependent Independent Acquisition (DIA) MS analysis (Library free and Library-based search):
28. Merge the direct-DIA search archive and DDA library to construct a hybrid library and use this library to perform library-based search of the DIA data.
SECTION: Database searches with Biognosys Spectronaut for Data Dependent Independent Acquisition (DIA) MS analysis (Library free and Library-based search):
29. Use the below settings for the library-based DIA search within Spectronaut.
 
     Spectronaut   15.7.220308.50606               Computer Name:   MRC-DRI-2               User Domain Name:   LIFESCI-AD               User Name:   rnirujogi               Analysis Mode: UI               Analysis Type:   Peptide-Centric               Settings Used: RN_DIA_Default               Data Extraction               MS1 Mass Tolerance   Strategy:      Dynamic        Correction Factor:      1        MS2 Mass Tolerance   Strategy:      Dynamic        Correction Factor:      1        Intensity   Extraction MS1:      Maximum Intensity        Intensity   Extraction MS2:      Maximum Intensity        XIC Extraction               XIC IM Extraction   Window:      Dynamic        Correction Factor:      1        XIC RT Extraction   Window:      Dynamic        Correction Factor:      1        Calibration               Calibration Mode:      Automatic        MS1 Mass Tolerance   Strategy:      System Default        MS2 Mass Tolerance   Strategy:      System Default        Precision iRT:      TRUE        iRT <-> RT   Regression Type:      Local (Non-Linear) Regression        Exclude Deamidated   Peptides:      TRUE        MZ Extraction   Strategy:      Maximum Intensity        Allow source   specific iRT Calibration:      TRUE        Used Biognosys' iRT   Kit:      TRUE        Calibration   Carry-Over:      FALSE        Identification               Generate Decoys:      TRUE        Decoy Limit   Strategy:      Dynamic        Library Size   Fraction:      0.1        Decoy Method:      Mutated        Preferred Fragment   Source:      NN Predicted Fragments        Machine Learning:      Per Run        Exclude Duplicate   Assays:      TRUE        Precursor PEP   Cutoff:      0.2        Protein Qvalue   Cutoff (Experiment):      0.01        Protein Qvalue   Cutoff (Run):      0.05        Exclude Single Hit   Proteins:      TRUE        Pvalue Estimator:      Kernel Density Estimator        Precursor Qvalue   Cutoff:      0.01        Single Hit Definition:      By Stripped Sequence        Quantification               Interference   Correction:      TRUE        MS1 Min:      2        MS2 Min:      3        Exclude All   Multi-Channel Interferences:      TRUE        Only Identified   Peptides:      TRUE        Protein LFQ Method:      Automatic        Major (Protein)   Grouping:      by Protein Group Id        Minor (Peptide)   Grouping:      by Stripped Sequence        Minor Group Top N:      TRUE        Min:      1        Max:      3        Minor Group   Quantity:      Mean precursor quantity        Major Group Top N:      TRUE        Min:      1        Max:      3        Major Group   Quantity:      Mean peptide quantity        Quantity MS-Level:      MS2        Quantity Type:      Area        Proteotypicity   Filter:      None        Data Filtering:      Qvalue        Cross Run   Normalization:      TRUE        Row Selection:      Automatic        Normalization   Strategy:      None        Normalization   Filter Type:      None        PTM Workflow               PTM Localization:      TRUE        Probability Cutoff:      0.75        PTM Analysis:      TRUE        Multiplicity:      TRUE        Run Clustering:      FALSE        PTM Consolidation:      Sum        Flanking Region:      7        Workflow               In-Silico Library   Optimization:      FALSE        Profiling Strategy:      iRT Profiling        Profiling Row   Selection:      Minimum Qvalue Row Selection        Qvalue Threshold:      0.01        Profiling Target   Selection:      Automatic Selection        Carry-over exact   Peak Boundaries:      FALSE        Unify Peptide Peaks   Strategy:      None        Multi-Channel   Workflow Definition:      From Library Annotation        Fallback Option:      Labeled        Protein Inference               Protein Inference   Workflow:      Automatic        Inference   Algorithm:      IDPicker        Post Analysis               Calculate Sample   Correlation Matrix:      TRUE        Calculate Explained   TIC:      None        Gene Ontology:      geneOntology/Ontologies\bgs_default_go-basic.obo        Differential   Abundance Grouping:      Major Group (Quantification Settings)        Smallest   Quantitative Unit:      Major Group (Quantification Settings)        Use All MS-Level   Quantities:      FALSE        Differential   Abundance Testing:      Un-Paired t-test        Assume Equal   Variance:      FALSE        Group-Wise Testing   Correction:      FALSE        Run Clustering:      TRUE        Distance Metric:      Manhattan Distance        Linkage Strategy:      Ward's Method        Z-score   transformation:      FALSE        Order Runs by   Clustering:      TRUE        Pipeline Mode               Post Analysis   Reports:               Scoring Histograms:      TRUE        Data Completeness   Bar Chart:      TRUE        Run Identifications   Bar Chart:      TRUE        CV Density Line   Chart:      TRUE        CVs Below X Bar   Chart:      TRUE        Generate SNE File:      TRUE        Store Iontraces in   SNE:      FALSE        Report Schema:      PTMSiteReport (Pivot), RN_PG_Pivot (Pivot), MSStats Report (v 3.7.3)   (Normal), Protein Quant (Normal), Protein Quant (Pivot), BGS Factory Report   (Normal)        Reporting Unit:      Across Experiment   
 
SECTION: Data analysis of DIA data and data visualization:
30. Export Protein group tables from Spectronaut in PG Pivot format.
SECTION: Data analysis of DIA data and data visualization:
31. For the Golgi-tag IP data, annotate using a complied list of know Golgi proteins from a resource e.g. (https://compartments.jensenlab.org/Search) and Uniprot-GO terms.
 

SECTION: Data analysis of DIA data and data visualization:
32. Prepare the data for differential analysis and this can be done using Perseus software suite (https://maxquant.net/perseus/). The basic functionalities of the software and various workflows can be adopted from the published literature (PMID: 27348712) and available tutorials (http://coxdocs.org/doku.php?id=perseus:start) on Youtube (https://www.youtube.com/c/MaxQuantChannel/featured)

SECTION: Data analysis of DIA data and data visualization:
33. Follow the Perseus workflow illustrated in Figure 2.
Figure: 2


 
SECTION: Data analysis of DIA data and data visualization:
34. The T-test results can be exported and could be analysed using other software suites such as curtain tool to visualize the volcano plot and associated protein raw intensities for all the conditions, protein domain architecture, STRING interaction prediction and alpha fold prediction.

SECTION: Data analysis of DIA data and data visualization:
35. Optional: In addition to using the Perseus other data quality can be done using custom R or Python Scripts (Provided below) and other relevant packages.

Figure: 1

 

SECTION: Scripts - R correlation plot
36. 
 

SECTION: Scripts - Python Network Interaction with Cytoscape and Plotly Dash
37.