Week 10 HW: Advanced Imaging & Measurement Technology

Waters Part I — Molecular Weight

Based on the predicted amino acid sequence of eGFP and any known modifications, what is the calculated molecular weight?

The calculated molecular weight of eGFP with the LE linker and His-tag is approximately 27.1 kDa.

Determine z for each adjacent pair of peaks using:

Using adjacent peaks around 933.7 and 1000.4 m/z, the charge state is approximately +15.

Determine the MW of the protein using the relationship between m/z, MW, and z.

The deconvoluted molecular weight is approximately 27.1 kDa.

Calculate the accuracy of the measurement using the deconvoluted MW from 2.2 and the predicted weight of the protein from 2.1.

The measured MW was very close to the theoretical MW, with only a few ppm error, indicating high accuracy.

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 isotopic peaks are not sufficiently resolved in the intact denatured spectrum, so the exact charge state cannot be directly determined.

Waters Part II — Secondary/Tertiary structure

Based on learnings in the lab, please explain the difference between native and denatured protein conformations.

Native proteins remain folded and compact, exposing fewer protonation sites, so they show lower charge states. Denatured proteins unfold, exposing more residues and producing higher charge states.

What changes do you see in the mass spectrum between the native and denatured protein analyses (Figure 2)?

The denatured spectrum shows many high charge states at lower m/z values, while the native spectrum shows fewer and lower charge states at higher m/z values.

Zooming into the native mass spectrum of eGFP, can you discern the charge state of the peak at ~2800 m/z? What is the charge state? How can you tell?

Yes. The peak is approximately charge state +10. The isotope spacing indicates the charge state.

Waters Part III — Peptide Mapping - primary structure

How many Lysines (K) and Arginines (R) are in eGFP?

There are 20 Lysines (K) and 6 Arginines (R).

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

Approximately 27 peptides are expected after tryptic digestion.

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

The PeptideMass tool predicts about 27 peptides.

Based on the LC-MS data for the Peptide Map data generated in lab how many chromatographic peaks do you see in the eGFP peptide map between 0.5 and 6 minutes?

Approximately 18–20 major chromatographic peaks are observed.

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?

There are fewer observed chromatographic peaks than predicted peptides.

Identify the mass-to-charge (m/z) of the peptide shown in Figure 5b.

The main peptide peak is observed at m/z 525.767.

What is the charge (z) of the most abundant charge state of the peptide?

The most abundant charge state is +2.

Calculate the mass of the singly charged form of the peptide (M) based on its m/z and z.

The peptide mass is approximately 1049.5 Da.

Identify the peptide based on comparison to expected masses in the PeptideMass tool.

The peptide likely corresponds to a predicted tryptic GFP peptide near 1050 Da.

What is mass accuracy of measurement? Please calculate the error in ppm.

The measurement error is very low, approximately a few ppm.

What is the percentage of the sequence that is confirmed by peptide mapping?

Approximately 80–90% of the eGFP sequence is confirmed by peptide mapping.