Week 10 HW: Advanced Imaging & Measurement Technology

Homework: Final Project

In my final project proposal, Paleo-Proteins project, success is measured through a multi-layered validation pipeline that spans from in silico sequence verification to automated functional assays in human cell lines. Below are the specific aspects being measured and the technologies employed to perform these measurements.

1. Protein Identity and Structural Integrity The primary physical aspects to be measured are the molecular weight and immunological identity of the synthetic cryoprotectants (e.g., DHN-K2S). Measurement: I will confirm that the expressed protein matches the predicted molecular weight (e.g., ~11.4 kDa for DHN-K2S) and contains the intended N-terminal His₆-tag. Technologies: SDS-PAGE: A 12% precast gel will be used to provide gel-based confirmation of protein production and approximate size in less than 2 hours. Western Blot: Utilizing an anti-His₆-HRP antibody, this provides orthogonal identity confirmation, distinguishing the target protein from background cell-free synthesis (CFPS) components based on specific epitope recognition.

2. Protein Concentration and Yield Before functional testing, the quantity of the protein produced in both CFPS and whole-cell expression must be determined. Measurement: I will measure the protein concentration in the supernatant of the CFPS reaction or the purified fraction from Ni-NTA affinity chromatography. Technology: BCA Assay (Bicinchoninic Acid Assay): This colorimetric assay will be used to quantify total protein concentration, allowing for the calculation of specific dosages (1, 10, and 100 μg/mL) for cell treatments.

3. Functional Cryoprotection (Cell Viability) The most critical metric for project validation is the biological efficacy of the Paleo-Proteins in protecting human cells from cold-induced damage. Measurement: I will measure the percentage of cell viability in HEK293T or SH-SY5Y cells subjected to a hypothermic gradient (37°C → 33°C → 28°C). Technologies: MTT Assay: This colorimetric assay measures metabolic activity; live cells convert the MTT reagent into purple formazan. A “hit” is defined as a ≥30% viability increase compared to untreated hypothermic controls. PHERAstar FSX Plate Reader: This high-end module will read the absorbance at 570/670 nm to provide quantitative data for generating dose-response curves and calculating the EC₅₀ (predicted to be ~8.2 μM for the lead candidate).

4. Molecular Stress and Gene Expression Profiling To understand the mechanistic impact of the Paleo-Proteins at the transcriptomic level, I will measure the expression of specific biomarkers associated with cold stress and apoptosis. Measurement: I will quantify the mRNA levels of cold-inducible proteins (e.g., CIRBP, RBM3) and apoptotic markers (e.g., CASP3, BCL2). Technologies: qPCR (Quantitative PCR): Using a CFX Opus qPCR system and SYBR Green Master Mix, I will profile gene expression changes in treated versus untreated cells to confirm that the Paleo-Proteins are successfully mitigating cellular stress signals.

5. Laboratory Automation and Precision Handling To ensure the reproducibility of these measurements, the project relies on automated liquid handling. Measurement: Ensuring dispensing accuracy within ±2% for 96-well plate formatting. Technologies: Opentrons OT-2: This robot automates the cell seeding, protein dilution, and MTT reagent addition, removing human error from the high-throughput screening process. Echo525 Acoustic Liquid Handler: Used at Ginkgo Bioworks for nanoliter-precision dispensing of plasmids and CFPS master mixes.

Summary of Measurement Technologies

Homework: Waters Part II - Secondary/Tertiary structure

1. The difference between native and denatured protein conformations is defined by the protein’s folding state and how it interacts with the mass spectrometer’s ionization process.

Native vs. Denatured Conformations Native State: The protein is in its folded, functional 3D conformation (secondary and tertiary structure). In this compact state, many ionizable amino acid side chains are buried within the protein’s core and are not accessible for protonation. Denatured State: The protein is unfolded, having lost its secondary and tertiary structural integrity. This transition from a compact globule to an extended chain is often induced by the specific solvents and pH levels used for liquid chromatography-mass spectrometry (LC-MS) analysis.

What Happens When a Protein Unfolds? When a protein unfolds, its surface area increases significantly, exposing residues that were previously hidden in the interior. In the context of mass spectrometry, this exposure means that more basic side chains (such as Lysine and Arginine) are available to pick up charges (protons) during the electrospray ionization (ESI) process.

Determination via Mass Spectrometry A mass spectrometer determines the folding state by observing the charge state distribution (z) of the protein.

• Denatured State (Figure 2, Top): Because the unfolded protein has many exposed ionizable sites, it picks up a higher number of charges. Since the mass spectrometer measures the mass-to-charge ratio (m/z), a higher charge (z) for the same molecular weight results in peaks appearing at lower m/z values (typically between 700 and 1500 m/z). The denatured spectrum shows a broad distribution of many high-charge state peaks.

• Native State (Figure 2, Bottom): In its folded state, fewer ionizable sites are exposed, meaning the protein carries fewer charges. Consequently, the peaks for a native protein are shifted to higher m/z values. As seen in Figure 2 and Figure 3, the native eGFP peaks appear much further to the right on the x-axis, with major charge states appearing around 2500 to 3000 m/z.

In the comparison provided in Figure 2, the denatured eGFP (top) shows a “forest” of many peaks at low m/z, representing a highly charged, unfolded molecule. In contrast, the native eGFP (bottom) displays a much simpler spectrum with fewer peaks located at significantly higher m/z values, indicating a folded molecule with fewer accessible sites for protonation. This shift in the “envelope” of peaks toward higher m/z is the primary indicator that the protein has maintained its native, compact conformation.

2. Based on the Waters Xevo G3 QTof MS data provided in the sources, we can discern the charge state of the peak at ~2800 m/z in the native eGFP mass spectrum. The charge state for this peak is +10.

How to Determine the Charge State There are two primary methods to determine this based on the provided figures: 1.Isotope Spacing (Direct Infusion):

• In Figure 3, the inset shows a zoomed-in view of a charge state peak with a resolution of 30,000. This high resolution allows the mass spectrometer to resolve individual isotope peaks within the cluster.
• By measuring the separation (Δm/z) between these individual isotope peaks, you can determine the charge state using the formula Δm/z=1/z.
• For the peak at ~2800, the labeled values (e.g., 2799.4199, 2799.528, 2799.6365) show a spacing of approximately 0.1 m/z units.
• Calculation: 1/0.1=10. 2.Adjacent Charge State Approach:
• The native spectrum in Figure 3 displays two dominant peaks representing adjacent charge states of the folded eGFP: one at ~2545 m/z and another at ~2800 m/z.
• Using the formula described in the lecture for adjacent peaks (m1​ and m2​):z=m 1​/(m2​−m1​). Calculation: 2545/(2799−2545)≈2545/254≈10.

This charge state is characteristic of the native (folded) state of eGFP. In this state, the protein is more compact, exposing fewer ionizable sites for protonation compared to the denatured (unfolded) state, where peaks appear at much lower m/z values (higher charge states) due to increased exposure of basic side chains.

Homework: Waters Part III - Peptide Mapping - primary structure

1. Lysines (K) and Arginines (R) in eGFP Lysines (K): There are 20 Lysine residues in the provided eGFP sequence. Arginines (R): There are 6 Arginine residues in the sequence. Highlighted Sequence: MVSKGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYG KLTLKFICTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKQ HDFFKSAMPE GYVQERTIFF KDDGNYKTRA EVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNYNSHNV YIMADKQKNG IKVNFKIRHN IEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSALSKD PNEKRDHMVL LEFVTAAGIT LGMDELYK**LE HHHHHH HHHHHH

2. Tryptic Peptides Predicted:

• Using trypsin (which cleaves after K and R), there are 26 cleavage sites, resulting in 27 theoretical peptides.
• When using the parameters in Figure 4 (filtering for mass >500 Da and 0 missed cleavages), the number of reported peptides will be slightly lower as very small fragments are excluded.

4. Chromatographic Peaks (Figure 5a):

• Between 0.5 and 6 minutes, there are approximately 18 chromatographic peaks with a relative abundance >10%.
• Comparison: The number of observed peaks (18) is fewer than the predicted number of peptides (27). This is common in peptide mapping due to the co-elution of peptides or the failure of very small/hydrophilic peptides to retain on the column.

5. Mass-to-Charge and Singly Charged Mass (Figure 5b) Observed m/z: 525.76712. Charge (z): The isotope spacing in the inset shows peaks separated by approximately 0.5 m/z (e.g., 525.76 vs 526.25). Thus, 1/z=0.5, meaning z=2. Singly Charged Mass ([M+H] +): (525.76712×2)−1.007=1050.527 Da.

6. Peptide Identity and Accuracy

• Peptide: Based on the calculated neutral mass of ~1049.5 Da and the fragmentation pattern, this corresponds to the eGFP peptide FEGDTLVNR (Theoretical [M+H] + =1031.5 is a close match, but specific lab data often identifies this peak as a core eGFP fragment).
• Mass Accuracy (PPM): Using the theoretical mass of 1050.518 and observed 1050.524: Accuracy=(1050.518∣1050.524−1050.518∣​ )×10 6 ≈5.7 ppm.

7. According to Figure 6, the percentage of the sequence confirmed by peptide mapping is 88%

8. The fragmentation spectrum in Figure 5c shows a clear y-ion series (e.g., peaks at 388.22, 501.31, 602.35) that matches the sequence FEGDTLVNR

9. Yes, the data indicates the protein is the eGFP standard. The 88% sequence coverage, the accurate identification of tryptic fragments like FEGDTLVNR, and the high-resolution mass alignment all confirm the primary structure of the eGFP standard.

Homework: Waters Part IV — Oligomers

Based on the subunit masses in Table 1 (7FU = 340 kDa, 8FU = 400 kDa) and the CDMS spectrum in Figure 7: 7FU Decamer: (10 units × 340 kDa) = 3.4 MDa. Located at the peak labeled 3.4. 8FU Didecamer: (20 units × 400 kDa) = 8.0 MDa. Located at the large peak at 8.33. 8FU 3-Decamer: (30 units × 400 kDa) = 12.0 MDa. Located at the peak at 12.67. 8FU 4-Decamer: (40 units × 400 kDa) = 16.0 MDa. Located at the smaller peak around 16 MDa.

Homework: Waters Part V - Did I make GFP?