Size-Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS)

SEC-MALS Instrumentation

SEC-MALS at the CMI

The CMI has a SEC-MALS system with a Wyatt Dawn Heleos Multi-Angle Light Scattering (MALS, 18-angle) Detector, with in-line DLS detector and Optilab TrEX refractive index detector.  SEC-MALS is used to measure the weight-averaged molar mass of proteins (and other macromolecules) in solution, and can determine oligomeric state and sample polydispersity.  Size-exclusion chromatography (SEC) separates molecules based on hydrodynamic volume, but is dependent on similarity to a set of reference standards for accurate mass determination and fails for elongated or sticky proteins.  Multi-Angle Light static Scattering (MALS) is used to measure light scattering intensity accurately, which is proportional to the weight-averaged mass in solution. Combining SEC, MALS and concentration detectors in an SEC-MALS experiment allows for more accurate mass measurements that SEC or MALS alone.

By using two concentration detectors (RI and UV), the molar mass and weight-fraction of a modifier can be determined. This can be used to measure protein and modifier masses of integral membrane proteins in detergent micelles and of glycosylated proteins.

 

 

wyatt helleos image              SEC-MALS Data -BSA

SEC-MALS System Components

MALS

  • Wyatt Dawn Heleos II Multi-Angle Light Scattering (MALS) detector
  • Wyatt Optilab T-rex Refractive Index Detector
  • Wyatt In-line QELS detector for Dynamic Light Scattering

Chromatography

  • Agilent 1260 Infinity Isocratic Liquid Chromatography System
  • Agilent 1260 Infinity Autosampler
  • inline solvent degasser
  • variable wavelength UV detector

There is no fraction collector on this system, as it is intended for analytical and not preparative chromatography.

SEC-MALS Column Data

Data Files - About CMI Data Files

Users are responsible for storage of all raw and processed data collected at the CMI.

  • Users should have a plan to copy or transfer all raw and process data to their own local or cloud storage system.
  • While the CMI allows temporary local storage of CMI User data on the instrument computer, we make no guarantees on the security or long-term availability of any data at the CMI.
  • For most (but not all) CMI technologies, the raw data files and recommended readable exports are relatively small and can be readily transferred electronically. 
  • See specific instruments for exceptions and for details about the software, data file types and recommended data exports. 

Data Sharing:

  • Currently, a Generalist Repository is the recommended data repository for most CMI data types, as stable specialist data repositories have not been established.

Data Files - SEC-MALS - Dawn Heleos II

Technology Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS)
Instrument Agilent 1260 Infinity LC System with variable UV detector
Wyatt Dawn Heleos II MALS detector
Wyatt Optilab T-rEX Refractive Index Detector 
Recommended Repository Generalist Repository
       
Software Type Data Collection & Analysis
Current Version Astra, Version 7.
Data Files (Type, ~size) experiment file .afe7 3-5 MB/measurement
Readable Exports basic collection .csv 4 MB/measurement
  report .pdf 300-350 KB/measurement
  easi graph .csv 5 MB/experiment
  easi table .csv 3-5 KB/experiment

Light Scattering Data Collection Services

Light Scattering Services Description

In addition to instrument training, the CMI is now offering basic Light Scattering Data Collection services, including:

Static Light Scattering: SEC-MALS or Mass Photometry

  • mass determination in solution
  • oligomeric state

Dynamic Light Scattering

  • hydrodynamic radius
  • buffer optimization

Mass Photometry Service Overview

Mass Photometry

CMI Refeyn Two MP

Standard Mass Photometry Service

For proteins and protein complexes at least 30 KDa, without high molecular weight modifiers (modifiers of mass >5% total).  

  • Detector calibration with your buffer using a protein standard
  • Basic Data Analysis, in triplicate, 
    • Mass for each major peak
    • Population distribution of major peaks

AAV Mass Photometry Services

For AAVs (replication-incompetent, ~3-5 MDa). Requires Empty Capsid control, provided by the user.

Empty/Full Service

  • Detector calibration with your buffer using a protein standard
  • Basic AAV Analysis, in triplicate,
    • Empty/Full population distribution

AAV Extended Service

  • Detector calibration with your buffer using a protein standard and a DNA standard on poly-lysine coated slides
  • Extended AAV Analysis, in triplicate,
    • Empty/Full population distribution
    • Mass calculated for DNA cargo

SEC-MALS Service Overview

Size-exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS)

CMI SEC-MALS System: Wyatt Dawn Heleos with in-line DLS, RI UV, Agilent chromatography

Standard SEC-MALS Service

for proteins and protein complexes between 15 KDa and 1 MDa.  Prior purification by size-exclusion chromatography (SEC) is strongly recommended.  

Includes:

  • Column equilibration with your buffer (pH at or below pH 7.5) or with PBS
  • BSA Standard to test column performance and normalize detectors
  • SEC separation of your protein sample(s)
  • Basic Data analysis, including:
    • Molar Mass for each major peak
    • Hydrodynamic Radius from DLS (when light scattering signal is sufficient)

Additional Services

  • Protein Conjugate Analysis
    • required for samples with high molecular weight modifiers, such as glycosylation or detergent micelle (modifiers of mass >5% total)
    • requires modifier dn/dc
  • dn/dc measurements of detergent or protein samples

DLS Service Overview

Dynamic Light Scattering (DLS)

CMI DLS Instrument: Wyatt Dyanpro Plate Reader III

 

Standard DLS Service

For proteins and biopolymers up to 1000 nm in diameter. Includes:

  • Cumulants and Regularization fit of Hydrodynamic Radius (Rh), 1 nm - 1000 nm
  • Polydispersity Analysis

Additional Services

  • Aggregation Analysis - Small molecule
  • Buffer optimization

 

Data Collection Fees Summary

Data Collection

  • Limited Data Collection Services are offered.
  • Service fees are based on labor and supplies costs, and will be charged for all completed services, regardless of experimental outcome. 
  • Before submitting samples for data collection, users must approve the estimated charges and be given a date and time for sample delivery.
    • External Users will also be required to submit a PO and a signed CMI User Agreement.
  • Most CMI Data Collection Services include a setup fee plus a per-sample data collection fee.
    • Some services include replicate measurements by default in the per-sample fee. For others, there is a reduced-price replicate measurement fee, if collected in the same dataset.
  • Nanobody services not available to commercial users at this time.
  • Current Harvard Life Lab commercial users are offered a 25% discount off the standard commercial rates.

All Data Collection Fees

Generally, SEC-MALS data collection is done in ASTRA 7 with HPLC control.  

CMI SEC-MALS Getting Started Guide

CMI SEC-MALS Guide to Protein Conjugate Analysis, guide to measuring dn/dc values and performing protein conjugate data analysis.

CMI SEC-MALS Guide to Working with Custom Solvents, for solvents that cannot use PBS or water as the solvent model.

SEC-MALS Technology page from Wyatt Technologies.

 

  • purified protein samples (must be filtered before loading)
  • running buffer (1L)
  • analytical SEC column (the CMI usually has 1-2 working SEC-MALS columns that users may borrow, if their samples are purified by SEC and in an appropriate buffer)

 

Assay Buffers

  • Running buffer should always be chosen to be compatible with both the SEC column and the protein sample.
  • Recommended Buffer: 25 mM HEPES pH 7-7.5, 150 mM NaCl (filtered).
  • Make sure you know the buffer compatibility of the SEC column you are using.
    • Most silica columns will not tolerate pH above 7.5.
  • Some buffer components (e.g. glycerol) will require customization of the solvent profile. Take note of the absolute refractive index of the solvent after equilibration and before data collection.

Samples

  • The sample should be prepared in running buffer to minimize RI peak due to sample solvent.
    • This is particularly important for samples that run near the solvent peak.
    • It is good practice to run a size-exclusion chromatography purification prior to analytical SEC-MALS to buffer exchange and clear aggregates. This is a requirement if you are using a shared CMI column.
  • Samples must be filtered or centrifuged prior to injection.
  • Concentration should be accurately measured to assess column recovery.
    • Know the concentration (in mg/ml) of your protein.
    • Know the UV extinction coefficient (in ml/mg•cm) of your protein
  • Protein aggregates can damage the column.
    • Filter or centrifuge samples before use.
    • Assess protein heterogeneity via dynamic light scattering (in the DynaPro plate reader).
    • Purify protein samples with soluble aggregates by size-exclusion chromatography.
  • Recommended protein concentration varies depending on protein mass:
    • Scattering is proportional to mass
      • Larger proteins require less sample than smaller proteins.
    • Typical range 5 – 500 µg/injection
      • BSA (67 KDa) 100 µl at 2 mg/ml always gives a good light scattering signal
  • Sample volume:
    • Maximum injection volume: 100 µl
    • Minimum injection volume: 5 µl
    • Glass autosampler vial with low volume insert has 10 µl dead volume.
      • Fill vial with at least 110 µl for a 100 µl injection.