Week 10 HW: Imaging and Measurement

Homework: Waters Part I — Molecular Weight

We were analyzing 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).

1. Based on the predicted amino acid sequence of eGFP (see below) and any known modifications, what is the calculated molecular weight?

The chemical formula and average molecular weight. Sequence Components:

eGFP Core: MVSKGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYG KLTLKFICTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKQ HDFFKSAMPE GYVQERTIFF KDDGNYKTRA EVKFEGDTLV NRIELKGIDF KEDGNILGHK LEYNYNSHNV YIMADKQKNG IKVNFKIRHN IEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSALSKD PNEKRDHMVL LEFVTAAGIT LGMDELYKLE Linker: LE Tag: HHHHHH

Using an average isotopic mass calculator (like ExPASy Compute pI/Mw), the breakdown for this specific sequence is:

Calculated MW: 28,124.08 Da

2. 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 (n, n+1) using the formula provided

The relationship between two adjacent peaks, where $m_1$ is the lower $m/z$ peak (higher charge) and $m_2$ is the higher $m/z$ peak (lower charge).

  • The formulas were a bit hard to understand.

  • So far I have the following in mind but not sure
  • Z=31
  • m/zn = 903
  • MW= 31 × 903.7148 − 31