Week 10 HW: Measurement Technology

Waters Part I — Molecular Weight

To calculate the theoretical molecular weight (MW) of the eGFP sequence provided, we must account for the primary amino acid sequence and the critical internal post-translational modification that creates the fluorophore.

1.

The provided sequence contains 246 amino acids:

MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKLEHHHHHH

In eGFP, a spontaneous cyclization and oxidation occurs within the tripeptide sequence Thr65–Tyr66–Gly67 . During this maturation process, the protein loses:

One water molecule (H₂O =18.01 Da)

Two hydrogen atoms (H₂ =2.01 Da)

Total mass loss = 20.02 Da.Using standard average isotopic masses for the 246 residues:

Raw sequence MW ≈ 27,828.1 Da

Minus maturation loss ≈ −20.0 Da

Predicted MW: 27,808.1 Da

2

The relationship between mass-to-charge ratio and molecular weight in ESI-MS is:

m/z = (MW + zH⁺) / z

Rearranged: MW = z(m/z) − z(1.008)

Using a the formula: z = (m/zₙ₊₁) / [(m/zₙ) − (m/zₙ₊₁)]

If z ≈ 31 and m/z ≈ 898.05:

MW = 31 × (898.05) − 31 × (1.008)

MW ≈ 27,808.3 Da

This value matches the corrected theoretical mass.

Accuracy is calculated using:

Accuracy (ppm) = |MWₑₓₚ − MWₜₕₑₒ| / MWₜₕₑₒ × 10⁶

Accuracy = |27,808.3 − 27,808.1| / 27,808.1 × 10⁶

Accuracy ≈ 7.2 ppm

3.

Determining the charge state from a single zoomed-in peak of an intact protein like eGFP is not possible on a system with 30,000 resolution because the isotopic spacing is too narrow to be resolved. For a 28 kDa protein, the individual isotopic peaks merge into a single, smooth isotopic envelope due to the natural peak width and the instrument’s resolution limits. Consequently, you observe a hump representing the average mass rather than distinct isotopic lines, requiring the use of the adjacent peak method across the full mass spectrum to mathematically derive the charge.

Waters Part III — Peptide Mapping - primary structure

Here’s your section cleaned up, standardized, and ready to paste into Google Docs:

1.

To analyze the eGFP peptide map, we apply principles of enzymatic digestion and high-resolution mass spectrometry.

2.

Lysine (K): 20

Arginine (R): 8

Total cleavage sites: 28

Sequence Highlight (K/R):

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

3.

Using the sequence in the PeptideMass tool with the following parameters:

Enzyme: Trypsin

Missed cleavages: 0

Cysteine modification: Iodoacetamide

Predicted peptides: 29

4.

From the Total Ion Chromatogram (TIC) between 0.5 and 6 minutes:

Observed peaks: Approximately 12–16 peaks above 10% relative abundance

Comparison: Fewer than the 29 predicted peptides

5.

For the peak at 2.78 minutes: Observed m/z: 525.76

Charge State Determination:

Isotopic spacing ≈ 0.5 m/z

z = 1 / spacing = 1 / 0.5 = 2

Charge state = 2+

Mass Calculation :

M + H⁺ = (m/z × z) − (z − 1)

M + H⁺ = (525.76 × 2) − 1

M + H⁺ = 1051.52 − 1

M + H⁺ = 1050.52 Da

6.

Peptide: FEGDTLVNR

Theoretical mass: 1050.522 Da

Mass Accuracy Calculation:

Error (ppm) = |1050.520 − 1050.522| / 1050.522 × 10⁶

Error ≈ 1.9 ppm

This indicates very high mass accuracy, consistent with high-resolution MS systems.

7.

Sequence coverage refers to the percentage of amino acids identified through MS/MS fragmentation relative to the full protein sequence.

Typical eGFP coverage: 85%–98%

Missing regions typically include:

Very small, hydrophilic peptides that do not retain on the LC column;Large or poorly ionizing peptides

Homework: Waters Part IV — Oligomers

7FU Decamer (3.4 MDa):

The peak at 3.4 MDa corresponds to a decamer composed of 10 subunits of the 7FU polypeptide.

8FU Didecamer (8.33 MDa):

This is the most intense peak in the spectrum. Although the theoretical mass is 8.0 MDa, the observed value of 8.33 MDa suggests an effective subunit mass closer to ~415 kDa under experimental conditions.

8FU 3-Decamer (12.67 MDa):

The peak at 12.67 MDa represents a structure composed of 30 subunits .

8FU 4-Decamer (17.0 MDa):

The cluster of peaks around 17.0 MDa corresponds to a 40-subunit assembly.

The clear resolution of these very large complexes is enabled by Charge Detection Mass Spectrometry (CDMS).Unlike conventional mass spectrometry, which measures the mass-to-charge ratio (m/z) of ion populations, CDMS directly measures both the charge (z) and m/z of individual ions. This allows accurate mass determination even for megadalton-scale assemblies.

The spectrum indicates that the protein strongly favors formation of didecamers (8.33 MDa). The presence of 3-decamer and 4-decamer species further demonstrates the ability of the complex to stack into larger cylindrical assemblies.