Novalix ASMS Kd Demo

The Novalix ASMS Kd Demo details Virscidian's standalone manual data workflow involving data acquisition, transfer, and analysis with Analytical Studio-Pro, specifying required and optional metadata formats for sample and assay information, alongside SMS+Express services that provide automated data management, analysis, and storage with APIs and sample list building for affinity selection mass spectrometry experiments.

Virscidian Environments

Standalone Manual Data Workflow

Data acquisition
Data transfer
Data analysis by Analytical Studio-Pro
Data output

Metadata Requirements (Standalone Environments)

Required Metadata:
- Workflow name: WN! (e.g., WN!KD-std)
- Assay type: AT! (e.g., AT!ASMS)
- Plate layout: PL! (e.g., PL!01TL,XY,NL,1+16,2+24)
- Sample location: L! (e.g., L!C2)
- Protein name: PROTNAME! (e.g., PROTNAME!Enzyme1)
- Protein concentration: PROTCONC! (e.g., PROTCONC!10)
- Protein concentration units: PROTCONCUNITS! (e.g., PROTCONCUNITS!uM)
- Note: Compound concentration metadata is stored in the targets file.
Nice to Have Metadata:
- Reaction time: RXNT! (e.g., RXNT!1)
- Reaction time units: RXNTUNITS! (e.g., RXNTUNITS!hour)
- Replicate: REP! (e.g., REP!1)

Example SI String (for comments section of injection sequence):

SI=WN!KD-STD:PL!01TL,XY,NL,1+16,2+24:L!B04:SAM!MSOnly:SCM!MSOnly:SUBCONC!0.041:SUBCONCUNITS!uM:SN!MSC2036175B-6:PROTNAME!Protein1:F!C22H24N6O4S:

Example of Sample Info Metadata Format

SI=AT!ASMS:L!A09:PL!01TL,XY,NL,1+8,2+12:BAR!ABC123
- SI denotes the start of a sample info string, followed by key-value pairs.
  1. 1.AT: assay type (e.g., ASMS for affinity selection MS)
  2. 2.L: well location (e.g., A09)
  3. 3.PL: plate layout (e.g., 01TL,XY,NL,1+8,2+12)
  4. 4.BAR: barcode for tracking (e.g., ABC123)

SMS + Express Sample Management Services

Analytical Studio-Express
- Metadata, sample info, results management
- Data analysis & processing
- APIs available
- Data storage on an MS SQL database server
- Automated data transfer by Instrument Agent
- Sample List Builder creates sample lists / work lists
- Data output acquisition
- Analytical Studio-Pro (injection sequences)

Metadata (for SMS+Express Environments)

Fields:
- ASMS campaign number
- Plate Barcode
- PLATE_TYPE
- Well
- Compound ID
- Formula
- Adduct Override
- Batch
- IS Flag
- Mixture
- RT (min)
- RT Window (min)
- Tracking ID
- Concentration
- Concentration Units
- Protein name
- Protein Concentration
- Protein Concentration Units
Example Data:
- Kd Demo 001, 384, B01, Cpd-001, C22H24N6O4S, 0, uM
- Kd Demo 001, 384, B04, Cpd-001, C22H24N6O4S, 0.041, uM
- Kd Demo 001, 384, B05, Cpd-001, C22H24N6O4S, 0.123, uM
- Kd Demo 001, 384, B06, Cpd-001, C22H24N6O4S, 0.37, uM
Metadata is mapped from CSV to SMS database.
Metadata is referenced by SMS and Express Databases.
Automated data transfer by Instrument Agent.
Sample List Builder creates sample lists / work lists.
Metadata written by SMS.

Kd Analysis

Experiment Setup

Plate layouts are shown for multiple compounds and concentrations.
Example: Compounds 1–5 distributed across plate rows and columns.

Kd Modeling

MS response is measured as peak area or peak intensity.
Bmax: Maximal MS response where all binding sites are occupied.
Kd: Compound concentration at half of Bmax.
Model fit equation:

y = (Bmax * x) / (Kd + x)
- y: MS response
- x: Compound concentration

Data Suitability: Curve Fitting

Curve fitting metrics:
- MS readout
- Bmax
- Kd
- R² (how well data fits the model)
Fit equation:

y = (Bmax * x) / (Kd + x)

Data Suitability: Linear Fitting

Linear fitting metrics:
- MS readout
- Bmax
- R² (fit to a line)
- Slope
- Y-Intercept
- Intercept to Bmax ratio
Linear fit equation:

y = (Bmax * x) / (Kd + x)

Intercept to Bmax Ratios

Values approaching 1: Not enough low concentration data points.
Values approaching 0 (or negative): Not enough high concentration data points or a non-specific Kd binder.