Luiza Niyazmetova, SST NU

Outline What is mass spectrometry?

How it works?

Types

Application

Protein characterization and sequencing

ESI

MALDI

AMS

Conclusion

References

Mass Spectrometry Analytical technique that is used to measure mass-to-

charge ratio of charged particles

How it works

How it works Sample is loaded onto the mass spectrometer, and

undergoes vaporization

Components of the sample are ionized (e.g., by impacting them with an electron beam)

Ions are separated according to their mass-to-charge ratio in analyzer by electromagnetic fields

Ions are detected by a quantitative method

Ion signal is processed into mass spectra

Types AMS (Accelerator Mass Spectrometer (MS)) or

Tandem MS

MALDI (matrix-assisted laser desorption/ionization source)

Electrospray ionization (ESI) MS

TOF (time-of-flight MS)

Inductively coupled plasma-mass spectrometry (ICP-MS)

Thermal ionization-mass spectrometry (TIMS)

Spark source mass spectrometry (SSMS)

Application Identifying unknown compounds

Determining the isotopic composition of elements in molecule

Determining the structure of a compound by observing its fragmentation

Respiratory gas analysis in hospitals

Space exploration (analysis of plasma)

Proteomics research (protein characterization & sequencing)

Protein characterization and sequencing

Protein sequencing - determining amino acid sequence & conformation that protein adopts and extent to which it is complexed with non-peptide molecules

Important for creating protein databases

Characterization and quality control of recombinant proteins (ESI, MALDI)

Protein identification (sequencing) (ESI, MALDI, AMS)

Detection and characterization of posttranslational modifications (AMS)

Electro-spray ionization Softest ionization technique

Best technique for polar non‐volatile compounds (proteins, peptides, nucleic acids, Pharmaceuticals, natural products)

Not very sensitive to non-volatile salts

Allows producing multiple charged ions and determining high molecular weight proteins

Electro Spray ionization Sample is dissolved in polar volatile buffer

It is dispersed by electro spray into fine aerosol

Aerosol is directed through regions of higher vacuum until droplets evaporate to near atomic size

Molecules are transferred into the mass spectrometer with high efficiency for analysis

Matrix-assisted laser desorption/ ionization (MALDI)

1. Desorption: triggered by UV laser beam

- Matrix material heavily absorbs UV laser light, leading to the ablation of upper layer of matrix material

- Hot plume produced during the ablation contains neutral and ionized matrix molecules, protonated & deprotonated matrix molecules, matrix clusters & nanodroplets

2. Ionization (protonation or deprotonation):

- Takes place in the hot plume

- Some ablated species participate in protonation (deprotonation) of analyte molecules

Tandem MS Consists of two analyzers:

quadrupole - quadrupole

magnetic sector - quadrupole

magnetic sector - magnetic sector

quadrupole - time-of-flight

One peptide species out of mixture is selected in the first mass spectrometer

Dissociated by collision with inert gas (argon or nitrogen)

Resulting fragments are separated in the second part of the tandem mass spectrometer, producing the tandem mass spectrum

Protein sequencing by Tandem Mass Spectrometry

Protein sequencing - determining amino acid sequence & conformation the protein adopts and extent to which it is complexed with non-peptide molecules

1. Proteases break protein into peptides 2. Tandem Mass Spectrometer breaks the peptides down into

fragment ions & measures the mass of each piece 3. NH-CH, CH-CO, and CO-NH bonds can fragment along

amino acid backbone 4. Each bond breakage gives rise to two species, one neutral

and the other one charged 5. Only Charged species is monitored by the mass

spectrometer

Conclusion Protein sequencing is used for creating protein

databases

Common Mass spectrometry techniques used for protein sequencing:

- ESI

- MALDI

- Tandem (AMS) mass specrtometry

References Ashcroft, A.E. (2008). An introduction to mass spectrometry.

Retrieved from http://www.astbury.leeds.ac.uk/facil/MStut/mstutorial.htm

Basics of mass spectrometry. (2012). Retrieved from http://masspec.scripps.edu/mshistory/whatisms_details.php#Basics

Protein sequencing and identification with mass spectrometry. (2004). Retrieved from http://cseweb.ucsd.edu/classes/wi05/cse206b/notes/L4.pdf

The mass spectrometer. (2000). Retrieved from http://www.chemguide.co.uk/analysis/masspec/howitworks.html