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Extending the span of angular-scanning surface plasmon resonance biosensing:
hyphenation, variable-wavelength excitation, and multiplexing Lakayan, D.
2018
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Lakayan, D. (2018). Extending the span of angular-scanning surface plasmon resonance biosensing:
hyphenation, variable-wavelength excitation, and multiplexing.
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Appendix A. Supplementary information
Chapter 02 44
Figure S1. Sensorgram of HSA protein immobilization in channel 1 (black line). After obtaining the baseline with a continuous buffer flow; (A) both channels were cleaned with a mixture of NaCl and NaOH;
(B) surfaces of both channels were activated with a mixture of NHS/EDC; (C) Ligand (HSA) was injected to the sample channel only (black line) while the reference channel kept the same flow without HSA analyte; (D) both channels were then deactivated with ethanolamine; E,F.) the surface of both channels was finally cleaned with regeneration buffer (NaOH) to be ready for analysis.
Figure S2. RPLC-MS analysis of (A) the antibody preparation and (B) the papain-digested antibody preparation.
Extracted ion chromatograms (EICs) of the observed protein species are shown at their indicated m/z value with spectral baseline subtraction. The provisional deconvoluted identity of the species is shown in Table S1.
Chapter 02 45
Figure S3. Fractions were taken for tryptic digestion followed by nanoLC-MS and Mascot database searching.
(A) Upper SEC chromatogram represents the “Before Papain digestion” SEC chromatogram (i.e.
the intact antibody preparation). The three different areas fractionated and subjected to tryptic digestion, MS analysis and database searching are represented in three color codes. The hits of the search results from Mascot are given in the same color code. (B) The same is done for the
“After Papain digestion” SEC chromatogram of a papain digested antibody preparation. In this case, ten fractions were analyzed and again (as shown with the dotted colored lines representing each fraction) the hits of the search results from Mascot are given in the same color code.
Chapter 02 46 Figure S4. SPR binding sensorgram of a triplicate injection of an intact antibody preparation after SEC separation.
Regeneration steps are shown after each chromatographic run (negative signals). The sample channel showed a high affinity for eluting anti-HSA where a very low binding rate was observed with the reference channel. Non-specific binding to the surface and bulk effects were monitored from the reference channel with the total signal from the sample channel yielding the actual specific SPR interaction on the sensor after subtraction (not shown in the figure).
Figure S5. UV calibration curve and SPR sensor chip affinity saturation curve (logarithmic scale) of the HSA antibody preparation measured using optimized SEC-SPR parameters.
Chapter 02 47
a. The mass spectrum shows glycosylation, which in IgG1 is commonly found on the Fc part of the antibody.
b. The mass spectrum is devoid of glycosylation, indicating that is most likely a Fab fragment.
Table S1. Main molecular weight calculated from the mass spectra obtained in the apices of the peaks shown in Figure S2. The provisional assignment is based on molecular weight only.
Table S2. The association rate constant (ka), the dissociation rate constant (kd) and the equilibrium dissociation constant (KD) for standalone SPR and for SEC-SPR analysis of the intact antibody.
Chapter 02 48
