Week 10 HW: Imaging & Measurement Technology

Week 10 — Imaging & Measurement Technology

Constantin Convalexius · Lifefabs Node · HTGAA 2026

Lecturers: Evan Daugharthy, Lindsay Morrison, and the Waters Corp. team

Final Project — What I Will Measure

For my final project, I am building a cell-free split-GFP biosensor for the PARP1 catalytic domain / HPF1 protein-protein interaction. The wet-lab system has three Twist-synthesized constructs in a pET-style T7-lacO-RBS vector:

  1. PARP1cat-WT-GFP11
  2. PARP1cat-E988K-GFP11
  3. HPF1-GFP1-10

The measurement plan touches several analytical layers, including fluorescence, intact mass, peptide mapping, and optional fold/quality-control assays.

What I Want to Measure

PropertyWhy it matters
Intact protein mass of all three constructsConfirms each protein expressed at approximately the predicted molecular weight in CFPS. Unexpected masses could mean truncation, frame shift, degradation, or off-target proteolysis.
E988K mutation verificationThe final project includes a single-residue PARP1 mutation, so the mutation should be verified directly if possible. Intact MS gives only about a -1 Da shift from E to K, too small to rely on for a ~43 kDa protein. Peptide mapping is the better tool.
PPI signal: PARP1cat ↔ HPF1This is the direct biosensor readout. Split-GFP fluorescence at approximately 488/520 nm reports whether GFP1-10 and GFP11 come together when PARP1 and HPF1 are co-expressed.
Fold integrity, optionalDifferential scanning fluorimetry (DSF) with SYPRO Orange could compare the melting temperature of WT and E988K constructs.
Sample purity / protein QCEcho-MS or LC-MS on a subset of wells could help confirm that fluorescence results are connected to the intended protein products.

How I Would Measure Each Layer

MeasurementTechnologyWhy this technology
Intact protein massWaters Xevo G3 QToF or similar intact LC-MSCharge-state envelopes can be deconvoluted to estimate molecular weight.
E988K verificationWaters BioAccord LC-MS peptide mapping after trypsin digestResolves the peptide containing residue 988 and can confirm the specific mutation more directly than intact mass.
PPI biosensor signalSpark plate reader, sfGFP filter, approximately 488/520 nmEndpoint fluorescence quantification in a 384-well plate.
Fold integrityDSF / SYPRO Orange thermal meltGives a simple melting temperature comparison between WT and mutant constructs.
Sample QCEcho Mass Spectrometry, if availableFast sample-prep-light QC from multi-well plates.
Structural predictionAlphaFold3, Boltz-2, or ColabFoldIn silico context for whether E988K is predicted to disturb the PARP1cat-HPF1 interface.

Waters Part I — Molecular Weight

Q1. Calculated Molecular Weight of the eGFP Standard

Sequence: His-tagged eGFP with LE linker, 245 amino acids.

MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTL
VTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLV
NRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLAD
HYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKLE
HHHHHH

Using ExPASy ProtParam:

  • Number of amino acids: 245
  • Calculated molecular weight: 27,988.97 Da, approximately 27.99 kDa
  • Theoretical pI: approximately 6.06

This value is my theoretical molecular weight for the rest of the assignment.

Q2. Molecular Weight From Adjacent Charge States

I selected two adjacent peaks from the intact-MS spectrum:

  • m/z1 = 875.4421, the more highly charged ion
  • m/z2 = 903.7148, the adjacent, less highly charged ion

2a. Determine the Charge State

For adjacent charge states:

$$ z = \frac{m/z_2 - 1.0078}{m/z_2 - m/z_1} $$

Substitution:

$$ z = \frac{903.7148 - 1.0078}{903.7148 - 875.4421} = \frac{902.7070}{28.2727} = 31.92 $$

Rounded to the nearest integer:

  • The 903.7148 peak is z = 31+
  • The 875.4421 peak is z = 32+

2b. Calculate Molecular Weight

Using the more highly charged peak, z = 32+:

$$ MW = z \cdot (m/z) - z \cdot 1.0078 $$ $$ MW = 32 \cdot 875.4421 - 32 \cdot 1.0078 = 28{,}014.15 - 32.25 = \mathbf{27{,}981.90\ \mathrm{Da}} $$

Cross-check from z = 31+:

$$ MW = 31 \cdot 903.7148 - 31 \cdot 1.0078 = 28{,}015.16 - 31.24 = \mathbf{27{,}983.92\ \mathrm{Da}} $$

The two estimates agree within approximately 2 Da, which supports the charge-state assignment.

2c. Accuracy

$$ \mathrm{Error}_{ppm} = \frac{\left|MW_{exp} - MW_{theory}\right|}{MW_{theory}} \cdot 10^6 $$ $$ \mathrm{Error}_{ppm} = \frac{\left|27{,}981.90 - 27{,}988.97\right|}{27{,}988.97} \cdot 10^6 = \frac{7.07}{27{,}988.97} \cdot 10^6 \approx \mathbf{252.6\ ppm} $$

This is above the nominal mass accuracy expected from a calibrated QToF instrument. A likely explanation is that I am estimating the mass manually from a broad denatured charge-state envelope rather than using fully deconvoluted instrument software.

Q3. Charge State of the Zoomed-In Peak

Individual isotopes are difficult to resolve at this charge state on this instrument.

For intact eGFP at z = 32+, adjacent isotope peaks would be separated by:

$$ \Delta(m/z) = \frac{1}{z} = \frac{1}{32} \approx \mathbf{0.031\ m/z} $$

At m/z approximately 875, an instrument resolution of 30,000 gives a minimum resolvable difference of:

$$ \Delta(m/z)_{min} = \frac{875}{30{,}000} \approx \mathbf{0.029\ m/z} $$

This is right at the edge. In practice, the isotope envelope blurs into a broad peak rather than a clean isotope ladder.

Waters Part II — Secondary / Tertiary Structure

Q1. Native vs. Denatured Conformations

A denatured protein has been unfolded by acid, heat, organic solvent, or another denaturing condition. The polypeptide chain becomes more extended, exposing more basic residues such as lysine, arginine, and histidine. During electrospray ionization, more exposed sites can accept protons, so denatured proteins usually carry more charges. This shifts the charge-state envelope to lower m/z values and spreads it across many charge states.

A native protein remains folded in its biological three-dimensional conformation. Fewer protonation sites are exposed, so the protein usually carries fewer charges. This shifts the charge-state envelope to higher m/z values and makes it narrower.

In the mass-spec spectrum of eGFP, the denatured spectrum shows a broad cluster of charge states in the lower m/z range. The native spectrum shows fewer charge states shifted to higher m/z. This shift is a standard MS readout for folded versus unfolded protein states.

Q2. Charge State of the Peak at Approximately 2800 m/z

The peak at approximately 2800 m/z corresponds to z = 10+.

In the zoomed native spectrum, the isotope spacing is approximately:

$$ \Delta(m/z) \approx 0.10 $$ $$ z = \frac{1}{\Delta(m/z)} = \frac{1}{0.10} = \mathbf{10} $$

Cross-check:

$$ m/z = \frac{MW + z \cdot m_H}{z} $$ $$ m/z = \frac{27{,}989 + 10 \cdot 1.0078}{10} \approx \mathbf{2{,}799.9} $$

This matches the observed peak at approximately 2800 m/z.

Waters Part III — Peptide Mapping

Q1. Lysines and Arginines in eGFP

Counting lysine and arginine residues in the 245 amino-acid eGFP sequence:

  • Lysines (K): 20
  • Arginines (R): 6
  • Total trypsin cleavage sites: 26

Trypsin cleaves C-terminal to lysine and arginine, except in some cases where the next residue is proline. A simple first-pass estimate is therefore up to 27 peptides.

Q2. Number of Predicted Tryptic Peptides

Using ExPASy PeptideMass with trypsin, zero missed cleavages, cysteine carbamidomethylation, and no methionine oxidation:

  • Predicted peptides: 27

Q3. Chromatographic Peaks Visible

Counting peaks above approximately 10% relative abundance in the eGFP total ion chromatogram:

  • Approximately 18 chromatographic peaks are visible.

Q4. Predicted Versus Observed Peak Count

There are fewer visible chromatographic peaks than predicted tryptic peptides. Three reasons explain this:

  1. Very small peptides may elute in the void volume and be hard to detect.
  2. Very hydrophobic or very hydrophilic peptides may elute poorly or outside the observed window.
  3. Several peptides may co-elute at the same retention time.

A longer LC gradient would spread peptides out and improve separation.

Q5. m/z and Charge of the Peptide in Figure 5b

  • Most abundant m/z peak: 525.76712
  • Isotope spacing: approximately 0.50 m/z
  • Therefore charge: z = 2+

The singly charged mass is:

$$ [M+H]^+ = z \cdot (m/z) - (z - 1) \cdot 1.0078 $$ $$ [M+H]^+ = 2 \cdot 525.76712 - 1.0078 = \mathbf{1{,}050.53\ \mathrm{Da}} $$

Q6. Peptide Identification and Accuracy

Comparing the observed mass to the PeptideMass output, the closest match is:

FEGDTLVNR

This is a 9-residue peptide from the central beta-barrel region of eGFP.

Using the theoretical value from the peptide list:

$$ \mathrm{Error}_{ppm} = \frac{\left|1{,}050.53 - 1{,}050.52\right|}{1{,}050.52} \cdot 10^6 \approx \mathbf{9.5\ ppm} $$

This is within the expected range for peptide-level LC-MS identification.

Q7. Sequence Coverage

From the peptide mapping figure:

  • Sequence coverage: approximately 88%

This is strong coverage for confirming the identity of the eGFP standard.

Bonus 1. Fragment Ion Analysis

Using the candidate peptide FEGDTLVNR, the observed fragmentation pattern can be interpreted with b- and y-ion series. The presence of multiple matching b and y ions supports the assignment. Therefore, both intact peptide mass and fragmentation point to the same peptide.

Bonus 2. Did We Make eGFP?

Yes. The evidence supports that the protein produced is eGFP:

  1. The intact mass is close to the theoretical 27,988.97 Da.
  2. The native-state charge envelope is consistent with a folded protein.
  3. Peptide mapping gives approximately 88% sequence coverage.
  4. Fragmentation supports the identified peptide sequence.

Waters Part IV — Oligomers: CDMS of KLH

Charge-detection mass spectrometry, or CDMS, can resolve very large megadalton-scale assemblies that are difficult for conventional QToF analysis.

Using Table 1 and the assembly peaks shown in the lab figure:

SpeciesCompositionPredicted massObserved mass
7FU decamer10 × 7FU subunit, approximately 340 kDa eachapproximately 3.4 MDaapproximately 3.4 MDa
8FU didecamer20 × 8FU subunit, approximately 400 kDa eachapproximately 8.0 MDaapproximately 8.33 MDa
8FU 3-decamer30 × 8FU subunit, approximately 400 kDa eachapproximately 12.0 MDaapproximately 12.67 MDa
8FU 4-decamer40 × 8FU subunit, approximately 400 kDa eachapproximately 16.0 MDaapproximately 16 MDa

Slight differences between predicted and observed mass can come from carbohydrate, copper coordination, salt adducts, or natural heterogeneity in a large glycoprotein assembly.

Waters Part V — Did I Make GFP?

MetricTheoreticalObservedPPM mass errorMolecular weight
Mass27,988.97 Da27,981.90 Da252.6 ppm27.99 kDa theoretical / 27.98 kDa observed

The observed mass is within approximately 7 Da of the theoretical mass. The ppm error is higher than ideal for a calibrated instrument, but the manual calculation is based on broad charge-state peaks. Combined with native-state behavior, peptide mapping, and high sequence coverage, the data support that the protein is eGFP.

Connection to My Final Project

Two points from this lab are directly important for my PARP1-HPF1 biosensor project.

First, E988K verification cannot rely on intact MS alone. The mass shift from glutamate to lysine is only about -0.95 Da on a protein around 43 kDa. That is too small to confidently resolve by intact QToF analysis. The better method is tryptic peptide mapping of the peptide spanning residue 988.

Second, protein identity and quality control matter before interpreting fluorescence. If I see a split-GFP signal, I need to know whether the intended proteins were actually expressed. Intact MS, peptide mapping, or Echo-MS on representative wells could help confirm that fluorescence is coming from the designed constructs rather than from degradation products or expression artifacts.

One correction to my own thinking: CDMS is excellent for megadalton-scale assemblies like KLH, but my PARP1cat-HPF1 biosensor complex is much smaller. For this project, native MS or LC-MS protein QC would be more appropriate than CDMS.

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