Date: 12 August, 2020

Dataset Title: Dataset (TIRF microscopy movies) for "Direct kinetic fingerprinting and digital counting of single protein molecules"

Dataset Creators: Tanmay Chatterjee, Achim Knappik, Erin Sandford, Muneesh Tewari, Sung Won Choi, William B. Strong, Evan P. Thrush, Kenneth J. Oh, Ning Liu, Nils G. Walter, Alexander Johnson-Buck

Dataset Contact: Alex Johnson-Buck, alebuck@umich.edu

Funding: This work was funded by aLight Sciences, LLC, and Bio-Rad Laboratories, Inc, a University of Michigan Rogel Cancer Center Fund for Discovery Grant in Cellular Cancer Biology Imaging Research (CCBIR), a Grand Challenge Award from the A. Alfred Taubman Medical Institute.  Antibodies, animal serum, and recombinant antigens (except IL-34) were provided by Bio-Rad Laboratories, Inc. 

Research Overview:
The sensitive measurement of specific protein biomarkers is important for medical diagnostics and research. However, existing methods for quantifying proteins use antibody probes that cannot distinguish between specific and nonspecific binding, limiting their sensitivity and specificity. This work establishes a method for distinguishing between specific binding to the target protein and nonspecific binding to assay surfaces using single-molecule kinetic measurements with dynamically binding probes. This is significant because it permits extremely sensitive protein measurements without requiring a high-affinity detection antibody or any washing steps, enabling streamlined and sensitive quantification of proteins even when no pair of high-quality, tightly binding antibodies is available.

Methodology:
SiMREPS experiments were performed using one of two Olympus IX-81 objective-type TIRF microscopes equipped with cellTIRF and z-drift control modules (Olympus IX2-ZDC2 or ASI CRISP). While data acquired with the two microscopes are functionally identical, all measurements in a series of replicates or comparison experiments were performed on the same microscope. Detection Fabs were excited in TIRF mode with a theoretical penetration depth of ~80 nm using a fiber-coupled diode laser (Coherent, Inc.: CUBE 640-100C, 100 mW, or OBIS 637nm LX, 100mW) with an incident light intensity of ~100 W/cm2, and fluorescence emission was detected using an EMCCD (Andor IXon 897, or Photometrics Evolve) with an exposure time of 250 or 500 ms, after passing through a dichroic mirror and emission filter (Chroma, ZT640rdc-UF2 and ET655LP-TRF). In some experiments, an objective heater (Bioptechs) was used to raise the observation temperature to as high as 37 °C (calibrated using the reference thermistor provided by the manufacturer for the specific sample cell geometry used in this study).

Files contained here: .ZIP archives containing raw .TIFF files from the TIRF microscopy experiments listed below.

1. Standard curves for all four antigens in this study:
   - "IL-6 standard curves.zip" (standard washing and wash-free protocols)
   - "IL-34 standard curves.zip" (standard washing protocol)
   - "PAI-1 standard curves.zip" (standard washing protocol)
   - "VEGF-A standard curves.zip" (standard washing protocol)

2. Measurement of endogenous antigens in serum specimens from CAR-T patients:
   - "Measurement of IL-34 in patient serum.zip" (Patient 6, Days 1, 2, 8, and 15 post-CAR-T infusion)
   - "Wash-free measurement of IL-6 in patient serum.zip":
      - Patient 2: pre-infusion Baseline + Days 0,1,3,9,11,14,15,17,24,31, and 37 post-infusion
      - Patient 6: pre-infusion Baseline + Days 0,1,2,5,6,7,8,9,13,14, and 15 post-infusion
      - Patient 12: pre-infusion Baseline + Days 0,1,2,3,8,9,10,11, and 14 post-infusion

Related publication:
Chatterjee, T., et al. Direct kinetic fingerprinting and digital counting of single protein molecules. Proc Natl Acad Sci USA, In Press.

Use and Access: 
This dataset is made available under a CC BY-NC 4.0 license (https://creativecommons.org/licenses/by-nc/4.0/).

To Cite Data: 
Tanmay Chatterjee, Achim Knappik, Erin Sandford, Muneesh Tewari, Sung Won Choi, William B. Strong, Evan P. Thrush, Kenneth J. Oh, Ning Liu, Nils G. Walter, Alexander Johnson-Buck (2020). Direct kinetic fingerprinting and digital counting of single protein molecules [dataset]. University of Michigan - Deep Blue. https://doi.org/10.7302/na5e-vt32