Week 10 HW: Imaging and Measurement

LCMS LCMS

Final Project

  1. Please identify at least one (ideally many) aspect(s) of your project that you will measure. It could be the mass or sequence of a protein, the presence, absence, or quantity of a biomarker, etc.
  • In my project, I will measure the fluorescence that indicate the intensity of the biomarker present. Additionally, after the production of miRNA, I will measure their molecular weight to make sure they are right.
  1. Please describe all of the elements you would like to measure, and furthermore describe how you will perform these measurements.
  • I will measure it using UV Spectrophotometer for fluorescence and LC-MS for molecular weight
  1. What are the technologies you will use (e.g., gel electrophoresis, DNA sequencing, mass spectrometry, etc.)? Describe in detail.
  • Mass spectrometry will be used mainly for molecular weight determination to get the chromatogram and make sure from it that the produced peaks are correct.

Homework: Waters Part I — Molecular Weight

  1. The calculated molecular weight based on the predicted eGFP amino acid sequence is:
  • Theoretical pI is 5.90
  • Theoretical molecular weight is 28006.60 Da
  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:
  • Determine z for each adjacent pair of peaks (n, n+1) using:
    • the selected two charge states are (m/zn+1) = 875.4421 and (m/zn) = 903.7148
    • therefore, the z = 30.96 = 31
  • Determine the MW of the protein using the relationship between m/zn, MW and z
    • MW = (n x m/zn) - n
    • n (number of charges) = 31
    • MW = (31 x 903.7148) - 31 = 27,984.1588
  • Calculate the accuracy of the measurement using the deconvoluted MW from 2.2 and the predicted weight of the protein from 2.1
    • Accuracy = ((MW experiment - MW theory) / MW theory) x 1,000,000 = ((27,984.1588 - 28006.60) / 28006.60) x 1,000,000 = 801.282 ppm
  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?
    • No, the zoomed picture is quite blurry

Homework: Waters Part III — Peptide Mapping - primary structure

  1. Highlight Lysines (K) and Arginines (R) amino acids in the eGFP sequence

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

  1. How many peptides will be generated from tryptic digestion of eGFP?
  • The number of peptides generated when using trypsin to perform the digest is 19 peptides img img
  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.
  • The number of chromatographic peaks in the eGFP peptide map between 0.5 and 6 mins are 21 labeled peaks
figure5a figure5a
  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?
  • No, the number of predicted peptides are lower than the number of peptide peaks in the chromatogram.
  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.
  • the m/z of the peptide shown is 525.76
  • since the peptides are separated by app. 0.5 m/z, therefore, the charge (z) of the peptide is 2
  • the mass of the singly charged form of the peptide ([M+H]+)
    • m/z = (M + nH) / n
    • 525.76 = (M + 2H)/2
    • 1051.52 = M + 2 (1.00727)
    • M = 1049.5
    • therefore, M+H = 1050.51
  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.
  • Theoretical MW (average mass): 1050.5214 and experiment Mw (monoisotopic mass): 1050.51
  • Accuracy = 10.85 ppm
  • The peptide sequence is FEGDTLVNR
  1. What is the percentage of the sequence that is confirmed by peptide mapping?
  • 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):
figure7 figure7
  • 7FU decamer = 7FU mass x 10 = 340 kDa x 10 = 3400 kDa = 3.4 MDa
  • 8FU didecamer = 8FU mass x 20 = 400 kDa x 20 = 8000 kDa = 8 MDa
  • 8FU 3-decamer = 8FU mass x 30 = 400 kDa x 30 = 12000 kDa = 12 MDa
  • 8FU 4-decamer = 8FU mass x 40 = 400 kDa x 40 = 16000 kDa = 16 MDa
figure7afterdetermination figure7afterdetermination

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.

TheoreticalObserved/measured on the Intact LC-MSPPM Mass Error
Molecular weight (kDa) of FEGDTLVNR1050.52141050.5110.85 ppm

Resources

  1. https://web.expasy.org/compute_pi/
  2. https://web.expasy.org/peptide_mass/