TITLE: NRTDP Top Down Standard SOP: Orbitrap Eclipse and Exploris
AUTHORS: Rafael Melani, Paul Thomas, Bryon Drown

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This standard protocol details how to make a standard mix of proteins for tracking LC-MS performance and illustrates how to configure an instrument for high-quality data collection.</div><div class = "text-block">This protocol was developed with support from the National Institute of General Medical Sciences P41GM108569 for the National Resource for Translational and Developmental Proteomics (NRTDP) based at Northwestern University. The authors thank Melinda Wojtkiewicz from the University of Nebraska Medical Center for helping us troubleshoot this method for use with the Exploris 480 in the Gundry Lab.</div></div>

[STEPS]
?. [Prepare Standard Mix]
Prepare   stocks of each protein standard in Optima water. (Aliquots can be stored at  )
-80 °C
?. [Prepare Standard Mix]
Prepare the following mixture (volumes shown from respective stock solutions):ProteinVolume (uL)Stock Concentration (pmol/uL)Amount Loaded on Column (pmol, 1X)Carbonic Anhydrase4025.70.64Myoglobin4043.91.09Trysinogen2519.60.49Ubiquitin2.55.50.14Total107.5
ProteinVolume (uL)Stock Concentration (pmol/uL)Amount Loaded on Column (pmol, 1X)Carbonic Anhydrase4025.70.64Myoglobin4043.91.09Trysinogen2519.60.49Ubiquitin2.55.50.14Total107.5
?. [Prepare Standard Mix]
Divide final mixture into   aliquots and store at
2.5 µl
-80 °C
?. [LC Parameters]
Prepare and condition a self-pack PLRP-S columnPacking Material: PLRP-S resin, 1000 Å pore size, 5 µm particle size (obtained from Agilent Technologies)Trap column: 2 cm bed length, 150 µm I.D.Analytical column: 20 cm bed length, 75 µm I.D.Nanospray Emitter: 15 µm PicoTip emitter, packed with 2 mm PLRP-S resin (P/N FS360-50-15-N-20-C12, New Objective)
?. [LC Parameters]
Inject  onto trap column and execute the following gradient.Solvent A: 95% Optima H2O, 5% Optima Acetonitrile, 0.2% MS-grade formic acidSolvent B: 5% Optima H2O, 95% Optima Acetonitrile, 0.2% MS-grade formic acidTrapping configuration: 3 µL/min flow rate (10 min. trap cycle, 55 °C)Analytical configuration: 0.3 µL/min flow rate (48 min. analytical gradient, 55 °C)Time (min.)% BCurve051050% in 10 min141510% in 4 min404530% in 26 min429550% in 2 min46950% in 4 min50590% in 4 min6050% in 10 min
6 µl
Time (min.)% BCurve051050% in 10 min141510% in 4 min404530% in 26 min429550% in 2 min46950% in 4 min50590% in 4 min6050% in 10 min
?. [Prepare Standard Mix]
When ready for acquisition, dilute one aliquot of TD STD in 100x vol. of Buffer A (95% Optima water, 5% Optima acetonitrile, 0.2% MS-grade formic acid), where 1x vol. is the intended injection volume (e.g.   for an intended injection volume of  ). This will ensure that the correct amount of each TD standard protein is present in each injection.Mix thoroughly by pipetting, then transfer to a clean autosampler vial. The standard is now ready for use.
[Buffer A]
6 µl
?. [MS Acquisition]
The mass spectrometer was operated in the following configuration:Positive and profile mode, RF Lens 30%, with 15.0 V source CID. “Intact Protein” on if available, “Low Pressure” selected if available, Default Charge State 15, “Advanced Peak Determination” on
?. [Data Interpretation Analysis]
Example Data
?. [Data Interpretation Analysis]
Data analysis methods:ProSight Lite: The software is available for free download at http://prosightlite.northwestern.edu/. A detailed protocol for the analysis of the NRTDP Top-Down Standard with Xtract and ProSight Lite can be found at https://link.springer.com/content/pdf/10.1007%2F978-1-4939-6783-4_18.pdfProSight PC 4.0: A “Standards” search database for high-throughput data analysis of the NRTDP Top-Down Standard with ProSight PC 4.0 is available for download here: http://proteinaceous.net/database-warehouse/NRTDP TDPortal: A custom workflow for high-throughput analysis of the NRTDP Top Down Standard is available on the TDPortal Quest-based, high-performance computing environment available through NRTDP and Northwestern University. User accounts can be requested at http://nrtdp.northwestern.edu/tdportalrequest/. A detailed protocol for data analysis on TDPortal by external users (NRTDP SOP_004) can be found at http://nrtdp.northwestern.edu/wp-content/uploads/2017/01/ExternalUserJan10.pdf
?. [Data Interpretation Analysis]
Longitudinal Data TrackingThe NRTDP recommends including the following metrics into longitudinal tracking of LC and MS performance:Peak Intensity: IT and FT MS1 peak intensity of ubiquitin, myoglobin, and carbonic anhydrasePeak Area (log): Chromatographic peak area (and retention time) of ubiquitin, myoglobin, and carbonic anhydraseFWHM: Full width at half maximum of ubiquitin, myoglobin, and carbonic anhydrase peaksInjection Time: MS1 and MS2 injection times for ubiquitin, myoglobin, and carbonic anhydraseP-score (-log): ubiquitin, myoglobin, and carbonic anhydrase obtained by low- or high-throughput data analysis Paying close attention to these parameters over time can help identify LC or MS issues before they become significant, thus reducing loss of important sample data. The NRTDP further recommends running at least three injections of the Top Down Standard before and after running experimental samples, as well as at least one injection of Top Down Standard every twenty-four hours. Evaluation of these standards on the fly will not only help detect LC or MS issues, but also provide confirmation that optimal performance is maintained when consistency and reproducibility are crucial.
?. [MS Acquisition]
Method Parameters:Experiment 1Master Scan: FTMS1Scan Range (m/z)600-2000(120k RP)Microscans1 (2 if no low-pressure mode)Full ScanMax Inject Time (ms)100Normal mass rangeMS1 AGC Target1.2e6 (300%)Scan Event 1: ddMSNScanActivation typeHCDScan rangeDefine m/z range350-2000(60k RP)Quadrupole ioslation ONIsolation width (m/z)3Top N 3 sec, ddNormalized Collision Energy28Isolation offset, OFFMicroscans1 (2 if no low-pressure mode)Supplemental activation OFFMax Inject Time (ms)600Charge Filter: 5 MS2 AGC Target5e5 (1000%)Experiment 2 (Eclipse Only)Master Scan: ITMS1Scan Range (m/z)600-2000Full ScanMicroscans20Rapid scan rateMax Inject Time (ms)10Normal mass rangeMS1 AGC Target3e4Dynamic Exclusion Settings (MS2):Repeat Count1Exclusion Duration (s)60Exclusion Mass Width (High/Low, m/z)0.5Exclude IsotopesTrue
Experiment 1Master Scan: FTMS1Scan Range (m/z)600-2000(120k RP)Microscans1 (2 if no low-pressure mode)Full ScanMax Inject Time (ms)100Normal mass rangeMS1 AGC Target1.2e6 (300%)Scan Event 1: ddMSNScanActivation typeHCDScan rangeDefine m/z range350-2000(60k RP)Quadrupole ioslation ONIsolation width (m/z)3Top N 3 sec, ddNormalized Collision Energy28Isolation offset, OFFMicroscans1 (2 if no low-pressure mode)Supplemental activation OFFMax Inject Time (ms)600Charge Filter: 5 MS2 AGC Target5e5 (1000%)Experiment 2 (Eclipse Only)Master Scan: ITMS1Scan Range (m/z)600-2000Full ScanMicroscans20Rapid scan rateMax Inject Time (ms)10Normal mass rangeMS1 AGC Target3e4
Repeat Count1Exclusion Duration (s)60Exclusion Mass Width (High/Low, m/z)0.5Exclude IsotopesTrue