Week 10 - Advanced Measurement & Measurement Technology

Class Assignment

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).

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).

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 for each adjacent pair of peaks using:

Determine the MW of the protein using the relationship between

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

Figure 1. Mass Spectrum of intact eGFP protein from the Waters Xevo G3 LC-MS (a mass spectrometer with 30,000 resolution) with individual charge state peaks labeled with $\frac{m}{z}$ values. Figure 1. Mass Spectrum of intact eGFP protein from the Waters Xevo G3 LC-MS (a mass spectrometer with 30,000 resolution) with individual charge state peaks labeled with

values.

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? Homework: Waters Part II — Secondary/Tertiary structure Assignees for this section MIT/Harvard students Optional but highly recommended Committed Listeners Optional but highly recommended We will analyze eGFP in its native, folded state and compare it to its denatured, unfolded state on a quadrupole time-of-flight MS. We will be doing MS-only analysis (no liquid chromatography, also known as “direct infusion” experiments) on the Waters Xevo G3-QToF MS.

Based on learnings in the lab, please explain the difference between native and denatured protein conformations. For example, what happens when a protein unfolds? How is that determined with a mass spectrometer? What changes do you see in the mass spectrum between the native and denatured protein analyses (Figure 2)? Figure 2. Comparison of the mass spectra between denatured (top) and native (bottom) eGFP standard on the Waters Xevo G3 QTof MS. Figure 2. Comparison of the mass spectra between denatured (top) and native (bottom) eGFP standard on the Waters Xevo G3 QTof MS.

Zooming into the native mass spectrum of eGFP from the Waters Xevo G3 QTof MS (see Figure 3), can you discern the charge state of the peak at ~2800

? What is the charge state? How can you tell? Figure 3. Native eGFP mass spectrum from the Waters Xevo G3 Q-Tof MS. The inset is a zoomed-in view of the charge state at ~2800 $\frac{m}{z}$ on a mass spectrometer with 30,000 resolution. Figure 3. Native eGFP mass spectrum from the Waters Xevo G3 Q-Tof MS. The inset is a zoomed-in view of the charge state at ~2800

on a mass spectrometer with 30,000 resolution.

Homework: Waters Part III — Peptide Mapping - primary structure Assignees for this section MIT/Harvard students Required Committed Listeners Required 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.

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). How many peptides will be generated from tryptic digestion of eGFP? Navigate to https://web.expasy.org/peptide_mass/ Copy/paste the sequence above into the input box in the PeptideMass tool to generate expected list of peptides. Use Figure 4 below as a guide for the relevant parameters to predict peptides from eGFP. Click “Perform the Cleavage” button in the PeptideMass tool and report the number of peptides generated when using trypsin to perform the digest.