WEEK 10

Measurements Final project

1. Types of measurements

Biosensor types To be confirmed after for class on biosensors

G protein–coupled receptors (GPCRs)
Bacterial two-component system histidine kinase receptors (e.g., EnvZ, NarX, PhoQ-type sensors)
Ligand-gated ion channels (LGICs) or transporters (e.g., GLUT transporters, engineered channels)

GLYCOGEN measurements Membrane proteins that can detect glycogen levels in the environment or on the cell surface are generally designed as engineered biosensors, as natural extracellular glycogen sensors are rare. Key proteins and strategies identified in research include:

Stbd1 (Starch-binding domain-containing protein 1): Stbd1 is a human protein featuring a Carbohydrate-Binding Module (CBM20) that binds specifically to glycogen and is targeted to membranes via an N-terminal hydrophobic sequence. It has been utilized to create a fusion protein, GYSC, which serves as a probe to detect glycogen in mammalian cells and muscle fibers.
Engineered Glycogen-Binding Probes (e.g., Patent Blue V): While not a traditional membrane protein, Patent Blue V (PBV) is a fluorescent probe that binds specifically to glycogen and can be used in complex environments, making it a powerful tool for monitoring extracellular glycogen.
AMPK β-Subunit: The carbohydrate-binding domain of the AMP-activated protein kinase (AMPK) β-subunit acts as a sensor for intracellular energy reserves by binding to glycogen. Although typically intracellular, this mechanism highlights the protein’s ability to sense glycogen levels.
Lectin-Based Biosensors: Lectins are a family of proteins with strong binding affinity to specific carbohydrates, including glycogen and other glycans, making them useful in designing biosensors to detect carbohydrate levels in various environments. These sensors are often targeted to the cell surface, allowing researchers to monitor extracellular glycogen levels or cell-surface glycogen-binding activities.

Membrane protein-based biosensors https://pmc.ncbi.nlm.nih.gov/articles/PMC5938585/

A generic method for fluorescence monitoring glycogen through patent blue V triggered supramolecular switching https://www.sciencedirect.com/science/article/abs/pii/S0925400522002726

A red fluorescent genetically encoded biosensor for in vivo imaging of extracellular l-lactate dynamics https://www.nature.com/articles/s41467-025-64484-x

Protein Targeting to Glycogen (PTG): A Promising Player in Glucose and Lipid Metabolism https://www.mdpi.com/2218-273X/12/12/1755

A yeast FRET biosensor enlightens cAMP signaling https://www.molbiolcell.org/doi/10.1091/mbc.E20-05-0319

Novel method for detection of glycogen in cells https://pmc.ncbi.nlm.nih.gov/articles/PMC5444244/

In vivo biochemistry: Applications for small molecule biosensors in plant biology https://pmc.ncbi.nlm.nih.gov/articles/PMC3679211/

ACID LACTIC measurements (Next step)

BIOLUMINESCENCE

Membrane proteins play a crucial role in bioluminescence, acting as transporters for substrates, anchoring luciferases for localized signaling, or as part of energy transfer complexe.

Key membrane proteins and related mechanisms include:

Oatp1 (Organic Anion Transporting Polypeptide 1): Identified as a plasma membrane transporter for D-luciferin. Expressing Oatp1 alongside luciferase significantly increases light output in vivo by facilitating substrate entry into cells.
PDGFR Transmembrane Domain: Used to anchor luciferase enzymes, such as dinoflagellate luciferase, to the plasma membrane to monitor cell surface expression kinetics.
HaloTag/NanoLuc Fusion Proteins: GPCRs (G protein-coupled receptors) are often fused with NanoLuc (a bright luciferase) and HaloTag (a self-labeling protein) to measure cell surface expression, trafficking, and interactions.
Antenna Proteins (e.g., Lumazine protein): While sometimes soluble, some antenna proteins are membrane-associated. They receive energy from the excited state of the luciferase-luciferin complex and shift the emission color.
Q-BOLT (Quenching Bioluminescent Voltage Indicator): A hybrid system using a HaloTag-NanoLuc fusion localized to the plasma membrane via a pDisplay sequence, which acts as a membrane potential reporter.
Luminopsins: Fusion proteins combining a luciferase with light-sensitive ion channels (like channelrhodopsin) on the membrane, enabling artificial light generation to control membrane voltage.

In marine organisms, these membrane proteins are essential for the high-intensity, controlled flashes of light often seen in deep-sea creatures, where localization of the reaction to the membrane maximizes light output efficiency.

Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications https://pmc.ncbi.nlm.nih.gov/articles/PMC9820413/

Bioluminescence Assay for Detecting Cell Surface Membrane Protein Expression https://pmc.ncbi.nlm.nih.gov/articles/PMC3064531/

2. Measurement methods

Quantifying bioluminescence involves measuring the photons emitted from the luciferase-substrate reaction. The two primary methods are luminometry (for bulk samples in vitro) and bioluminescence imaging (for spatial distribution in cells or whole organisms).

Reference: Quantitative Analysis of Bioluminescence Optical Signal

3. Measurement technologies

Mass Spectrometry

1. Calculation of eGFP molecular weight

1a. Online calculator

Reference eGFP sequence:

MVSKGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYG KLTLKFICTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKQ HDFFKSAMPE GYVQERTIFF KDDGNYKTRA EVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNYNSHNV YIMADKQKNG IKVNFKIRHN IEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSALSKD PNEKRDHMVL LEFVTAAGIT LGMDELYKLE HHHHHH

According to Gemini:

In experimental settings, enhanced GFP (eGFP) is expressed with the start codon (N-terminal Methionine) as well as the His-purification tag (HHHHHH) and its linker (LE).
For the calculation, one needs to calculate the summation of all the residues (linear Mw, that includes the addition of an extra hydrogen in N-terminal and an extra OH in C-term) and take into account the chromophore maturation (self-catalyzed backbone cyclization and oxidation of the tripeptide Thr65–Tyr66–Gly67 that allows eGFP to become fluorescent, see diagram below), which leads to the loss of one water molecule (-18.02 Da) during cyclization and the loss of two hydrogen atoms (-2Da) during oxidation.
Average molecular weight is a best choice for the calculation because takes into account the natural distribution of all isotopes while monoisotopic weight only considers the single most abundant isotope for each atom. In simple words, the average estimation is closer to the natural distribution of isotopes present in nature.

Schematic diagram of the chromophore formation in maturing eGFP:

Source image: What is the maturation time for fluorescent proteins?

Calculation

When entering the full sequence in the online calculator one obtains a theoretical general molecular weight of ~ 28.006 kDa (“Th.Av. Mw” = 28006.60 Da)

Integration of the fluorophore maturation: Th.Av. Mw - (H2O Mw + H2 Mw) = 28006.60 - (18.02 + 2) = 28006.60 - 20.02 = 27986.58 Da

In conclusion, the molecular weight of eGFP is estimated to 27.987 kDa.

1b. Adjacent charge state approach

Pair of adjacent peaks selected from the intact LC-MS data:

M/Zn+1 = 875.4421 and M/Zn = 903.7148

According to the adjacent charge state formula presented during recitation:

n = [ (M/Zn+1) -1 ] / [ (M/Zn) - (M/Zn+1) ]

n = (875.4421 -1) / (903.7148 - 875.4421) = 874.4421 / 28.2727 = 30.9289

Thus, charge state adjacent peaks:

Zn = ~31+ and Zn+1 = ~32+

According to molecular weight formula presented during recitation:

MW = ( n x M/Zn ) - n

MW (31+) = ( 31 x 903.7148 ) - 31 = 28015.1588 - 31 = 27984.1588 Da

MW (32+) = ( 32 x 875.4421 ) - 32 = 28014.1472 - 32 = 27982.1472 Da

Thus, average experimental molecular weight:

MW = 27.983 kDa

According to the mass accuracy formula presented during recitation:

Accuracy = | MW experiment - MW theory | / MW theory x 1’000’000

With MW experiment = 27’983 Da and MW theory = 27’987 Da

Accuracy = | 27983 - 27987 | / 27987 x 1’000’000 = 142.92 ppm

Conclusion: If accuracy > 50 ppm, either the protein is denatured or the mass spectrometer was not calibrated.

1c. Charge state zoomed-in peak

The values of the zoomed-in peak are not readable.

2. Secondary/tertiary structure

2a. Charge-state distribution

Analyze of eGFP in its native, folded state and comparison with its denatured, unfolded state on a quadrupole time-of-flight MS (lab experiment on Waters Xevo G3-QToF MS).

When a protein gets denatured, it loses the compact 3D (tertiary) structure of its native form. This unfolding process increases the protein surface exposed to the solvent, which acquires more charges during electrospray ionization. The extended shape of the protein also increases the drift time through the tube.

The mass spectrometer can detect this by measuring the protein’s charge-state distribution:

Reference: Lecture Week 10 by Lindsay Morrison

Image credit: Waters Corporation (slides from the lecture)

2a. Charge-state ~800 m/z peak

According to the formulas above, Zn = (MW + n) / M/Zn

With MW_theory = 27,986.57 Da and M/Zn = 2799.4199 for the peak ~800 M/Zn.

Thus, Zn = 27986.57 / 2799.4199 = 9.9973, i.e. Zn = ~ 10+

3. Peptide mapping: primary structure

3a. Lysine (K) and Arginine (R) residues count

Manual count:

Lysine (K) = 20

Arginine (R) = 6

Results confirmed when analysing the peptide in Benchling

3b. Trypsin-generated peptides

Online tool used to predict the list of peptides generated from a tryptic digest: Expasy

Trypsin cuts after the Lysine (K) and Arginine (R) residues. If the digestion is complete, one can expect it to generate 27 (20+6+1) peptides.

However, when running the digestion in Expasy, the list only contains 19 peptides:

This discrepancy is explained by the application of a filter: PeptideMass only displays peptides above 500 Da.

So in conclusion:

Theoretical digest: 27 peptides
PeptideMass list (>500Da) = 19 peptides

3c. Peptide Map

Peaks count:

If the threshold is set relatively to the baseline intensity (10% above 5e^6): 21 to 23 peaks (depending if including unlabelled peaks or not)
If the threshold is set relatively to the max peak intensity (10% of 1.2e7): 18 peaks

3d. Experiment vs theory

PeptideMass predicted 19 peptides (>500 Da) but the experimental data are slightly different (18-23 peptides, depending on threshold applied). The degree of mismatch appears reasonable and might be explained by:

Peptides too small to be detected
Miscleavage
Sample degradation or modification (e.g. oxidation)
Peaks merging due to similar elution time
Impurities

3e. Analysis of the peptide 2.78 min retention time

Charge State

The most abundant charge state for the peptide 2.78 min retention time is M/Z = 525.76712

According to formula [M/Z_adj - M/Z] = [M_adj -M] / Z and given that adjacent peak M/Z_adj = 526.25918,

M/Z_adj - M/Z= 526.25918 - 525.76712 = 0.4921

Thus, Z = 1 / 0.4921 = 2.03 = ~2+

Mass of the singly charged form of the peptide [M+H]+

[M+H]+ = Z x (M/Z) - (Z-1) x H

with M/Z = 525.767, Z = 2 and H+ = 1.00728 Da

[M+H]+ = 2 x 525.76712 - 1 x 1.00728 = 1050.52691 Da

This result match the singly charged peak m/z = 1050.52438 that can be observable in Fig.5c:

3f. Peptide identification & Mass Accuracy

Match with PeptideMass generated list (see above): Peptide FEGDTLVNR 1050.5214 Da (residues 115–123)

Accuracy: Accuracy = | MW experiment - MW theory | / MW theory x 1’000’000

With MW experiment = 1050.52438 Da and MW theory = 1050.5214 Da

Accuracy = | 1050.52438 - 1050.5214 | / 1050.5214 x 1’000’000 = 2.837 ppm

3g. Peptide map coverage