SILAC(Proteomics)

Madhurima Saha

Proteomics

• Proteome is the entire complement of proteins, including the modifications made to a particular set of proteins, produced by an organism or system.

• Proteomics: Large-scale study of proteins, particularly their structures and functions. Mass spectrometry techniques are used.

Why study the proteome?

• Disparity exists between transcriptome and proteome- Amount of proteome = amount of transcriptome

• Transcriptome does not provide functional information- Script vs actor

• Proteome is more complex than genome and transcriptome- ~ 30,000 genes, 100,000 +transcripts, but 1,000,000+

unique proteins- PTMs

Mass Spectromerty (MS)• Mass Spectrometry (MS) is an analytical

technique that sorts ions based on their mass and charge.

Calutron mass spectrometers were used in the Manhattan Project for uranium enrichment.

LTQ Orbitrap

Mass Spectrometry Protocol

Tandem Mass Spectrometer• A tandem mass spectrometer is one capable of multiple rounds of mass

spectrometry, usually separated by some form of molecule fragmentation. • For example, one mass analyzer can isolate one peptide from many

entering a mass spectrometer. • A second mass analyzer then stabilizes the peptide ions while they collide

with a gas, causing them to fragment by collision-induced dissociation (CID).

• A third mass analyzer then sorts the fragments produced from the peptides. Tandem MS can also be done in a single mass analyzer over time, as in a quadrupole ion trap. An important application using tandem mass spectrometry is in protein identification

• MALDI-TOF, which simply refers to combining a matrix-assisted laser desorption/ionization source with a time-of-flight mass analyzer

MS vs MS/MS

Mass to Charge ratio (m/z)• A mass spectrum (plural spectra) is a plot of the ion

signal as a function of the mass-to-charge ratio. • These spectra are used to determine the elemental

or isotopic signature of a sample, the masses of particles and of molecules, and to elucidate the chemical structures of molecules, such as peptides and other chemical compounds.

• Mass spectrometry works by ionizing chemical compounds to generate charged molecules or molecule fragments and measuring their mass-to-charge ratios.

Chromatograph (RAW data)

Mass Spectrometry Protocol

b ions and y ions

Quantitative Proteomics

Quantitative proteomics methods can be classified into two broad categories—• methods based on stable isotope labeling that

introduce a mass difference between two proteomes OR

• provide an internal standard for relative quantification, and approaches that quantify proteomes using the integrated MS signal of each peptide.

Ong, S.-E. and M. Mann (2007). "A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC)." Nat. Protocols 1(6): 2650-2660.

Stable Isotope Labeling

The stable isotope labeling methods are further subdivided into two classes: • Chemical methods that utilize a reagent to

introduce the mass tag (iTRAQ) OR • Metabolic labeling methods that utilize

biological incorporation of stable isotope labels into living cells (SILAC).

Ong, S.-E. and M. Mann (2007). "A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC)." Nat. Protocols 1(6): 2650-2660.

iTRAQ• Isobaric tags for relative and absolute

quantitation (iTRAQ) is an isobaric labeling method used in quantitative proteomics by tandem mass spectrometry to determine the amount of proteins from different sources in a single experiment.

• It uses stable isotope labeled molecules that can be covalent bonded to the N- terminus and side chain amines of proteins.

SILAC• SILAC (stable isotope labeling by/with amino

acids in cell culture) is a technique based on mass spectrometry that detects differences in protein abundance among samples using non-radioactive isotopic labeling. It is a popular method for quantitative proteomics.

Isotopes• Isotopes are variants of a particular chemical

element which differ in neutron number, although all isotopes of a given element have the same number of protons in each atom.

For example, Carbon has 3 isotopes, Atomic number of Carbon =6carbon-12, Protons= ? Neutrons = ?

carbon-13 Protons= ? Neutrons = ?

carbon-14 Protons= ? Neutrons = ?

6

6

6

7

8

6

Stable Isotopes vs Radioactive Isotopes

• Some isotopes are radioactive, and are therefore described as radioisotopes.

• Others have never been observed to undergo radioactive decay and are described as stable isotopes.

• For example, 14C is a radioactive form of carbon (C) while 12C and 13C are stable isotopes.

• Nitrogen (N) consists of two stable isotopes, 14N and 15N. 13N is radioactive

What is the difference of m/z between light and heavy amino acids?

Heavy Arginine (C13 and N15)

m/z = ? 5

Heavy Lysine (C13 and N15)

m/z = ? 4

z = 2 z = 2m = 10 m = 8

Light(C12 and N14)

SILAC

WHY SILAC?1. Relative protein abundance information.2. The rate of synthesis and degradation of

each protein.3. Unique way to measure the protein turnover

at specific conditions.4. Wide picture of a dynamic measure of

protein flux in the cellular proteome.

Reagents NeededReagents• Cell line of choice (Fibroblasts)• Custom-synthesized cell culture medium without arginine, lysine,

(available from custom culture medium distributor; should be formulated identically to the routine cell culture medium but lacking arginine, lysine)

• Dialyzed fetal bovine serum (Invitrogen, cat. no. 26400-044)• Antibiotics (penicillin and streptomycin 100, Invitrogen)• Glutamine (100, Invitrogen)• L-Arginine C13 and N15 (Heavy)and L-Arginine C12 and N14 (Light)• L-Lysine C13 and N15 (Heavy)and L-Lysine C12 and N14 (Light)• Proline • Leucine

• Metabolic interconversion of Arg and Pro in certain cell lines. - Arg to Pro conversion usually occurs when an excess of arginine is provided to cells in growth medium.

• Heavy and Light mixing of media.• Growth of the cells are much slower because

of the dialyzed FBS.

Things to consider….