LABEL-BASED PROTEIN QUANTFICATION iTRAQ, TMT, SILAC introduction of itraq, tmt and silac iTRAQ Isobaric Tags for Rela�ve and Absolute Quan�ta�on (iTRAQ) technology u�lizes isobaric reagents to label the primary amines of pep�des and proteins. The iTRAQ reagents usually consist of an N-methyl piperazine reporter group, a balance group, and an N-hydroxy succinimide ester group that is reac�ve with the primary amines of pep�des. The balance groups present in each of the iTRAQ reagents func�on to make the labeled pep�des from each sample isobaric and the quan�fica�on is facilitated through analysis of reporter groups that are generated upon fragmenta�on in the mass spectrometer.
Amine specific pep�de reac�ve group(NHS)
Report Ion
Balancer Group
There are currently two mainly used reagents: 4-plex and 8-plex, which can be used to label all pep�des from different samples/treatments.
TMT Mass Reporter
Protein Reac�ve Group
Tandem Mass Tags (TMT) can be used to iden�fy and quan�fy proteins in different kinds of samples. Each TMT tagging reagent is composed of an amine-reac�ve NHS-ester group, a spacer arm and an MS/MS reporter. And the isobaric tagging reagent within a set has the same nominal parent (precursor) mass.
Mass Normalizer
Eleven tags are available in TMT to label almost any pep�de or protein samples. Using TMT, mul�plex analysis, �me efficiency, and control over technical varia�ons can all be achieved.
SILAC Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) technique performs protein labeling by adding light, medium, or heavy stable isotope-labeled essen�al amino acids (lysine and arginine) to cell culture media. Through the normal metabolism of the cell, a�er 5-6 mul�plica�on cycles, the stable isotope-labeled amino acids are completely incorporated into the newly synthesized proteins of the cell, so that the newly synthesized proteins are labeled with stable isotopes. In this way, the area size of isotope peak type in mass spectrogram was compared for rela�ve quan�fica�on, and the sequence of pep�de was determined by secondary harmonic diagram for protein iden�fica�on.
workflow of itraq, tmt and silac iTRAQ Pep�des
iTRAQ Labelling 114
191
PRG
115
190
PRG
116
189
PRG
117
188
PRG
Proteins
SILAC
Pep�des
iTRAQ Labelling Grow Cells in Medium with Light Isotopes
TMT10-126
TMT10-127N
TMT10-128C
Lysate Cells to Extract Proteins
TMT10-129N
Labeled Pep�de Mixture
TMT10-129C
TMT10-130N
Combine Lysates 1:1 Frac�onate
HPLC Separa�on of Pep�de Mixture TMT -130C 10
TMT10-131
MALDI MS/MS Analysis
Trypsin Diges�on
Intensity
HPLC Separa�on of Pep�de Mixture
Intensity
116 115
LC-MS/MS
Rela�ve Intensity
MALDI MS/MS Analysis m/z
Intensity
Treated
TMT10-128N
Mix
114
Grow Cells in Medium with Heavy Isotopes
Control
TMT10-127C
Mix
Proteins
TMT
8 Da
m/z
117
Intensity
m/z
NC
NC NC
NC
m/z
characteristics of itraq, tmt and silac Characteristics
iTRAQ
TMT
SILAC
Labeling Method
In vitro
In vitro
In vivo
Label Specifica�ons
4plex/8plex
Duplex/6plex/10plex
-
Repor�ng group molecular mass
113-121; The minimum molecular mass difference between labeled reagents was 1 Da
126-131; The minimum molecular mass difference between labeled reagents was 6/1000 Da
-
TOF and Orbitrap mass spectrometry
The mass spectrometer has a resolu�on of at least 50,000 to dis�nguish different tags. Older versions of obtrap instruments can only dis�nguish 6plex
TOF and Orbitrap mass spectrometry
High throughput High protein coverage Quan�ta�ve accuracy High credibility Data-rich
A. Labeling efficiency can be up to 100% B. Good quan�ta�ve repeatability, low protein consump�on C. Suitable for the iden�fica�on and quan�fica�on of membrane proteins D. Closer to the real state of the sample
Suitable mass spectrometer
Advantages
Disadvantages
High throughput High protein coverage Quan�ta�ve accuracy High credibility Data-rich
A. Sample prepara�on and enzyma�c processes may cause discrepancies between parallel samples, resul�ng in biased results. B. Occurrence of interference between co-screening and co-fragmenta�on of the precursor ion for complex samples.
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It is mainly suitable for passageable cells or bacteria. Other experimental materials are not applicable. Long turnaround �me