Danica Riza V. Delgado

IPMB-1 Practicals

Dec. 23, 2010

Institute of Molecular Biology andBiotechnology

Proteomics“Proteomics is a science thatfocuses on the study of proteins :their roles, their structures, theirlocalization, their interactions, andother factors.”

www.lexicon-biology.com

What is mass spectrophotometry?

- analytical tool used

for measuring the molecular mass of a molecule by measuring the mass-to-charge ratio (m/z) of its ion.

Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI

TOF) mass spectrometer

Electrospray Ionization mass spectrometer (ESI-MS)

Mass Spectrometers

LC/LC-MS/MS-Tandem LC, Tandem MS

Detection

Deflection

Accelaration

ionization

Ionizer

Sample

+_

Mass Analyzer Detector

MALDI

Electrospray

(Proteins must be charged and dry)

Create ions Separate ions Detect ions

• Mass spectrum

• Database analysis

MALDI-TOF

MW

Triple Quadrapole

AA seq

MALDI-QqTOF

AA seq and MW

QqTOF

AA seq and protein modif.

Requires point of plane to occur

Ionization Methods

Electrospray Ionization

Ionization Methods

MALDI

337 nm UV laser

cyano-hydroxycinnamic acid

++

+

+

-

-

-

++

+

+

-

-

--++

Analyte

Matrix

Laser

+

++

+

+

+Sample Target for

MALDI

MALDI-Time of Flight (TOF) Mass Spectrometry

Protocol

All proteins are sorted based on a

mass to charge ratio (m/z)

m/z ratio:

Molecular weight divided by the charge on this protein

Interpretation of Results

Typical Mass SpectrumRelative

Abundance

120 m/z-for singly charged ion this is the mass

MALDI-TOF Mass Spectrum of Insulin and b -lactoglobulin. A mixture of 5 pmol each of insulin(I) and b -lactoglobulin (L) was ionized by MALDI, which produces predominately singly chargedmolecular ions from peptides and proteins (I + H+ for insulin and L + H+ for lactoglobulin). However,molecules with multiple charges as well as small quantities of a singly charged dimer of insulin, (2 I + H)+,also are produced. [After J. T. Watson, Introduction to Mass Spectrometry, 3d ed. (Lippincott-Raven, 1997),p. 282.]

Methods for protein

identification

Applications Biotechnology: the analysis of proteins,

peptides, oligonucleotides

Pharmaceutical: drug discovery,

combinatorial chemistry, pharmacokinetics, drug metabolism

Clinical: neonatal screening, haemoglobin analysis, drug testing

Environmental: PAHs, PCBs, water quality, food contamination

Geological: oil composition

Applications Accurate molecular weight measurements

Reaction monitoring

Amino acid sequencing

Oligonucleotide sequencing

Protein structure

References:

• Bauer Core Standard Protocol (http://sysbio.harvard.edu/csb/resources/downloads/Bauer_Core_Opticon2_Protocol.doc)

•Mass spectrometry-based proteomics. Aebersold R and Mann M. Nature. VOL 422, 13 March 2003

•Stryer, L., Berg, J., and Tymoczko, J. 2002. Biochemistry. 5th ed. New York: W H Freeman

• Ashcroft, Alison E. An Introduction to Mass Spectrometry (http://www.astbury.leeds.ac.uk/facil/MStut/mstutorial.htm)

•http://en.wikibooks.org/wiki/Proteomics/Protein_Identification_-_Mass_Spectrometry