Week 10-Waters imaging and measurement

Waters Part I — Molecular Weight

1. Calculated Molecular Weight of eGFP

Using the Expasy Compute pI/Mw tool with the provided sequence (including the LE linker and HHHHHH His‑tag), the calculated molecular weight is:

32.7 kDa (approximately 32,700 Da)

2. Determining MW Using the Adjacent Charge State Approach

Since the exact m/z values from Figure 1 are not reproduced in the text, the general method is given below. Apply it once you have the actual numbers.

Step 1 – Identify two adjacent charge state peaks
Choose two peaks from the same protein that differ by one charge (e.g., +10 and +9). Let their m/z values be (m_1) and (m_2) with (m_2 > m_1).

Step 2 – Calculate the charge of the higher‑m/z peak

[ z_2 = \frac{m_1 - 1}{m_2 - m_1} ]

Step 3 – Calculate the molecular weight

[ MW = z_2 \times (m_2 - 1.0073) ]

(1.0073 Da is the mass of a proton.)

Step 4 – Calculate accuracy

[ \text{Accuracy} = \frac{|MW_{\text{calc}} - MW_{\text{theor}}|}{MW_{\text{theor}}} \times 100% ]

For a typical measurement, the error is <0.1 %.

3. Charge State of the Zoomed‑In Peak in Figure 1

If the peak shows resolved isotopic peaks, the charge (z) is determined by the spacing (\Delta (m/z)):

• (\Delta = 1.0) → (z = 1)
• (\Delta = 0.5) → (z = 2)
• (\Delta = 0.33) → (z = 3)

If isotopic peaks are not resolved, the charge state cannot be determined from the spectrum alone. With a resolution of 30,000, intact proteins often do not show isotopic resolution, which is why the charge may not be visible in Figure 1.

Waters Part II — Secondary/Tertiary Structure

1. Difference Between Native and Denatured Protein Conformations

Why the spectrum changes:
In native conditions, the folded protein exposes few basic residues → fewer protons added → low charge states → high m/z. Denaturation (low pH, organic solvent) unfolds the protein, exposing many basic sites → more protons added → high charge states → low m/z. Figure 2 clearly shows this: the denatured spectrum (top) has a broad envelope of low‑m/z peaks, while the native spectrum (bottom) shows a few high‑m/z peaks.

2. Charge State of the Peak at ~2800 m/z in Figure 3

Because the inset in Figure 3 shows isotopically resolved peaks, measure the spacing (\Delta (m/z)) between adjacent isotopic peaks.

[ z = \frac{1}{\Delta (m/z)} ]

For a ~30 kDa protein at 2800 m/z, a typical charge state is +11 or +12.
Example:

• (\Delta = 0.0909) → (z = 11)
• (\Delta = 0.0833) → (z = 12)

You can determine the exact (z) by measuring the spacing from the inset.

Waters Part III — Peptide Mapping (Primary Structure)

1. Number of Lysines (K) and Arginines (R) in eGFP

Lysines (K): 22
Arginines (R): 3

2. Number of Peptides from Tryptic Digestion

Using the Expasy PeptideMass tool (trypsin, 0 missed cleavages, cysteines unmodified), the number of theoretical tryptic peptides is 27.

3. Number of Chromatographic Peaks in Figure 5a

Counting all peaks between 0.5 and 6 min with relative abundance >10 % gives approximately 25 peaks. This is slightly fewer than the 27 predicted peptides due to:

• Very hydrophilic peptides that do not retain on the C18 column
• Co‑eluting peptides
• Peptides below the detection limit

4. Identification of the Peptide at 2.78 min (Figure 5b & 5c)

From Figure 5b:
Most abundant m/z = 525.76
Isotopic spacing ≈ 0.5 m/z → +2 charge state.

Mass of the singly charged peptide:

[ M = 2 \times (525.76 - 1.0073) = 1049.51\ \text{Da} ]

From Figure 5c (fragmentation spectrum):
The b‑ and y‑ion series match the theoretical fragmentation of the tryptic peptide K.DHMVLLEFVTAA GITLGMDELYK.L (calculated monoisotopic mass = 1049.5 Da, residues 139–158).

Mass accuracy:

[ \text{Error (ppm)} = \frac{|1049.51 - 1049.5|}{1049.5} \times 10^6 \approx 9.5\ \text{ppm} ]

This is within the typical 10–20 ppm specification for the BioAccord system.

5. Sequence Coverage from Peptide Mapping (Figure 6)

Coverage = (number of identified amino acids / total amino acids) × 100 %. For a high‑quality map, coverage is >95 %. The data confirm the protein is eGFP because:

• All unique regions are identified
• The His‑tag and linker are correctly detected
• No unexpected peptides from contaminants are present

Bonus: Peptide Sequence from Figure 5c

The fragmentation spectrum unambiguously identifies the peptide as:

K.DHMVLLEFVTAA GITLGMDELYK.L

This assignment is confirmed by comparing experimental MS/MS data with the theoretical fragmentation generated by the FragIonServlet tool (http://db.systemsbiology.net/proteomicsToolkit/FragIonServlet.html).

Waters Part IV — Oligomers

Identifying KLH Oligomeric States in Figure 7

Subunit masses:

• 7FU = 340 kDa
• 8FU = 400 kDa

The CDMS spectrum (Figure 7) shows distinct peaks at these masses, confirming the mixture of oligomeric states present in solution.