Week 10 HW: Week 10 — Advanced Imaging & Measurement Technology

Homework: Final Project Measurement Draft

The main aspect of my project that I will measure is the functional activity of a mutated InaZ construct, specifically whether it increases ice nucleation efficiency relative to a control InaZ construct. I will perform this measurement using a controlled freezing assay in which replicate samples are cooled gradually and monitored for the onset of ice formation. The primary data collected will be the temperature at which freezing begins in each sample. Ice formation will be detected through optical observation of crystal formation and through temperature sensors that record the freezing point. In addition, I would verify the identity of the mutated construct using PCR, gel electrophoresis, and DNA sequencing. PCR and gel electrophoresis would be used to confirm the presence and approximate size of the inserted DNA, while DNA sequencing would confirm that the engineered inaZ mutation is correct. Together, these measurements allow me to confirm both that the construct was built properly and that it produces the intended increase in ice nucleation activity.

Restated:

Functional Assay: Ice Nucleation

Objective: Measure efficiency relative to a control construct.

Method: Controlled freezing assay with gradual cooling of replicate samples.

Data Points: Freezing onset temperature, recorded via combined optical observation and thermal sensors.

Genetic Verification

PCR & Gel Electrophoresis: Confirm the presence and approximate size of the inserted DNA.

DNA Sequencing: Verify the exact sequence of the engineered inaZ mutation.

Homework: Waters Part I — Molecular Weight

We will analyze an eGFP standard on a Waters Xevo G3 QTof MS system to determine the molecular weight of intact eGFP and observe its charge state distribution in the native and denatured (unfolded) states. The conditions for LC-MS analysis of intact protein cause it to unfold and be detected in its denatured form (due to the solvents and pH used for analysis).

  1. Based on the predicted amino acid sequence of eGFP (see below) and any known modifications, what is the calculated molecular weight? You can use an online calculator like the one at https://web.expasy.org/compute_pi/ 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 Note: This contains a His-purification tag (HHHHHH) and a linker (the LE before it).

Using the calculator, I get Theoretical pI/Mw: 5.90 / 28006.60.

  1. Calculate the molecular weight of the eGFP using the adjacent charge state approach described in the recitation. Select two charge states from the intact LC-MS data (Figure 1) and:
    1. Determine z or each adjacent pair of peaks (n , n+1) using: z = ((m/z_n+1)/(m/z_n) - (m/z_n+1)) = ((m/z_n+1)/(m/z_n) - (m/z_n+1))

z = ((966.0390)/(1000.4302-966.0390)= 28.09

z is roughly 28 (28+)

    1. Determine the MW of the protein using the relationship between MW = z (m/z-1.0073) = 28 (1000.4302-1.0073) = 27.98384 kDa
    1. Calculate the accuracy of the measurement using the deconvoluted MW from 2.2 and the predicted weight of the protein from 2.1 using:

Accuracy = (|Mw_Exp-ME_The|/Mw_The) = 27.98384-28006.60/28006.60 = |-22.75| 22.76 / 28006.60 = 0.0008126656 x 100 = 0.0812665586 =

  1. Can you observe the charge state for the zoomed-in peak in the mass spectrum for the intact eGFP? If yes, what is it? If no, why not?

Potentially. Assuming our peak as truly 1473.7420, our z is rougly 19+. I may need more information to answer more conclusively.

Homework: Waters Part II — Secondary/Tertiary structure

This was optional and skipped.

Homework: Waters Part III — Peptide Mapping - primary structure

We will digest the eGFP protein standard into peptides using trypsin (an enzyme that selectively cleaves the peptide bond after Lysine (K) and Arginine (R) residues. The resulting peptides will be analyzed on the Waters BioAccord LC-MS to measure their molecular weights and fragmented to confirm the amino acid sequence within each peptide – generating a “peptide map”. This process is used to confirm the primary structure of the protein.

There are a variety of tools available online to calculate protein molecular weight and predict a list of peptides generated from a tryptic digest. We will be using tools within the online resource Expasy (the bioinformatics resource portal of the Swiss Institute of Bioinformatics (SIB)) to predict a list of tryptic peptides from eGFP.

  1. How many Lysines (K) and Arginines (R) are in eGFP? Please circle or highlight them in the eGFP sequence given in Waters Part I question 1 above. (Note: adding the sequence to Benchling as an amino acid file and clicking biochemical properties tab will show you a count for each amino acid).

MVS[K]GEELFTG VVPILVELDG DVNGH[K]FSVS GEGEGDATYG [K]LTL[K]FICTT G[K]LPVPWPTL VTTLTYGVQC FS[R]YPDHM[K]Q HDFF[K]SAMPE GYVQE[R]TIFF [K]DDGNY[K]T[R]A EV[K]FEGDTLV N[R]IEL[K]GIDF [K]EDGNILGH[K] LEYNYNSHNV YIMAD[K]Q[K]NG I[K]VNF[K]I[R]HN IEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSALS[K]D PNE[K][R]DHMVL LEFVTAAGIT LGMDELY[K]LE HHHHHH

I counted 20 lysines and 6 arginine Residues.

  1. How many peptides will be generated from tryptic digestion of eGFP?

27 (26 + 1)

Done

    1. Copy/paste the sequence above into the input box in the PeptideMass tool to generate expected list of peptides.

Done

    1. Use Figure 4 below as a guide for the relevant parameters to predict peptides from eGFP.

Done

    1. Click “Perform the Cleavage” button in the PeptideMass tool and report the number of peptides generated when using trypsin to perform the digest.

Done

  1. Based on the LC-MS data for the Peptide Map data generated in lab (please use Figure 5a as a reference) how many chromatographic peaks do you see in the eGFP peptide map between 0.5 and 6 minutes? You may count all peaks that are >10% relative abundance.

I see between 18 and 27.

  1. Assuming all the peaks are peptides, does the number of peaks match the number of peptides predicted from question 2 above? Are there more peaks in the chromatogram or fewer?

Yes, at the upper end

  1. Identify the mass-to-charge (m/z)) of the peptide shown in Figure 5b. What is the charge (z)) of the most abundant charge state of the peptide (use the separation of the isotopes to determine the charge state). Calculate the mass of the singly charged form of the peptide [M+H]+) based on its m/z and z.

A) 525.76712 B) (2*525.76712)-(1.0073)= 1,051.53424 Da

  1. Identify the peptide based on comparison to expected masses in the PeptideMass tool. What is mass accuracy of measurement? Please calculate the error in ppm. (Recall that Accuracy = |MW experiment - MWtheory/MWtheory).

  2. What is the percentage of the sequence that is confirmed by peptide mapping? (see Figure 6).

Chain 1 = 88%

Homework: Waters Part IV — Oligomers

We will determine Keyhole Limpet Hemocyanin (KLH)’s oligomeric states using charge detection mass spectrometry (CDMS). CDMS single-particle measurements of KLH allow us to make direct mass measurements to determine what oligomeric states (that is, how many protein subunits combine) are present in solution. Using the known masses of the polypeptide subunits (Table 1) for KLH, identify where the following oligomeric species are on the spectrum shown below from the CDMS (Figure 7):

  • 7FU Decamer = (340 kDa x 10) = 3.4 MDa
  • 8FU Didecamer = (400 kDa x 20) = 8 MDa
  • 8FU 3-Decamer = (400 kDa x 30) = 12 MDa
  • 8FU 4-Decamer = (400 kDa x 30) = 16 MDa

Homework: Waters Part V — Did I make GFP?

Please fill out this table with the data you acquired from the lab work done at the Waters Immerse Lab in Cambridge, or else the data screenshots in this document if you were unable to have lab work done at Waters.

Screenshots listed on lab page.