## Basic principle of mass spectrometry

### 1. History questions:

- Who invented mass spectrometer?

*Joseph J. Thomson* - Who was the first one to win the Nobel Prize using mass spectrometry?

*Joseph J. Thomson (1906 Physics, electron)* - Who were the ones that discovered two modern methods for ionization of biological molecules for biomedical applications by mass spectrometry?

*John Fenn*: ESI;*Koichi Tanaka*: MALDI

### 2. Here is a part of a mass spectrum of a peptide ion:

- What is on the X-axial?

**mass/charge ratio**(m/z)

m = mass in Daltons (Da) or atomic mass units (u)

z = net positive or negative charge - What is on the Y-axial?

**the intensity of the ion signal at each particular m/z value** - Which peak is the monoisotopic ion?

**The first peak labeled with 558.7740** - What is the peak labeled with 559.2756?

~~The average ion~~**The [M+1] peak in Isotopic Ion Cluster (one atom altered)**

### 3. Using the same spectrum above, please calculate the charge state of the peptide ion.

$$

\begin{aligned}

\Delta\left(\frac{m}{z}\right)&=\frac{\left|558.7740-559.2756\right|+\left|559.2756-559.7764\right|+\left|559.2779-560.2779\right|}{3}\\ &=\frac{560.2779-558.7740}{3}\\ &=0.5013\approx\frac{1}{2}

\end{aligned}

$$

\begin{aligned}

\Delta\left(\frac{m}{z}\right)&=\frac{\left|558.7740-559.2756\right|+\left|559.2756-559.7764\right|+\left|559.2779-560.2779\right|}{3}\\ &=\frac{560.2779-558.7740}{3}\\ &=0.5013\approx\frac{1}{2}

\end{aligned}

$$

**So the answer is 2.**

### 4. What is the mass accuracy in ppm of the following peptide measurement (theoretical monoisotopic [M+H]+ = 1761.954)?

$$

ac={10}^6\times\frac{1761.954-1761.9211}{1761.954}=18.7ppm

$$

### 5. Calculate the mass of the intact protein from its ESI charge envelope shown below.

**Import data into matlab for fitting:**

$$

\frac{m}{z}=\frac{M}{n}+1

$$$$

x=n,y=\frac{m}{z}

$$

$$

M=52348

$$

## Mass spectrometry: Instrumentation

### 1. What are the solvents for ESI? How to improve solvent evaporation in ESI?

**Many solvents can be used in ESI and are chosen based on the solubility of the compound of interest, the volatility of the solvent and the solvent’s ability to donate a proton.**

- Heat: 250-300℃
- Neutral gas flow (nitrogen)
- Vacuum
- Volatile organic solvents: Methanol/Acetonitrile

### 2. If you need to choose a compound as the matrix to analyze an intact protein in MALDI, which one do you need to choose?

**UV light- absorbing weak organic acids**

### 3. What is the purpose of delayed extraction in MALDI?

**To reduce spreading of the ions and differences in initial velocity and energy so that ions of the same m/z travel together through the mass spectrometer and give high resolution peaks.**

### 4. There are many mass analyzers to choose from for a mass spectrometric analysis. Which factors do you need to consider for a mass analyzer?

**Speed(scan speed):**how fast an analyzer can complete an analysis over a specified mass range**Sensitivity:**represented by the lowest concentration of sample that can be detected by the instrument**Mass accuracy:**the difference between the measured and theoretical masses of an ion**Resolution:**the ability of the instrument to distinguish between ions of different m/z**Mass range:**the range of m/z values that can be effectively separated by the mass analyzer

### 5. Can you list at least five mass analyzers? Separate them into low and high resolution groups.

high | low |
---|---|

TOF FT-ICR Orbitrap |
Quadrupole Ion Trap |

### 6. For each mass analyzer, there is a relationship between an electronic parameter (voltage, currency, or frequency) and m/z of molecules. Can you derive its relation for TOF?

$$

\begin{cases}

\frac{1}{2}mv^2=Vze\\

\frac{d}{t}=v

\end{cases}

$$

\begin{cases}

\frac{1}{2}mv^2=Vze\\

\frac{d}{t}=v

\end{cases}

$$

$$

{\Rightarrow}\frac{m}{z}=\frac{2Vet^2}{d^2}

$$