Method for Quantitative Proteomics Research by Using Metal Element Chelated Tags Coupled with Mass Spectrometry

Abstract

The mass spectrometry-based methods with a stable isotope as the internal standard in quantitative proteomics have been developed quickly in recent years. But the use of some stable isotope reagents is limited by the relative high price and synthetic difficulties. We have developed a new method for quantitative proteomics research by using metal element chelated tags (MECT) coupled with mass spectrometry. The bicyclic anhydride diethylenetriamine-N,N,N',N' ',N' '-pentaacetic acid (DTPA) is covalently coupled to primary amines of peptides, and the ligand is then chelated to the rare earth metals Y and Tb. The tagged peptides are mixed and analyzed by LC-ESI-MS/MS. Peptides are quantified by measuring the relative signal intensities for the Y and Tb tag pairs in MS, which permits the quantitation of the original proteins generating the corresponding peptides. The protein is then identified by the corresponding peptide sequence from its MS/MS spectrum. The MECT method was evaluated by using standard proteins as model sample. The experimental results showed that metal chelate-tagged peptides chromatographically coeluted successfully during the reversed-phase LC analysis. The relative quantitation results were accurate for proteins using MECT. DTPA modification of the N-terminal of peptides promoted cleaner fragmentation (only y-series ions) in mass spectrometry and improved the confidence level of protein identification. The MECT strategy provides a simple, rapid, and economical alternative to current mass tagging technologies available.

ICP‐MS‐Based strategies for protein quantification

“…Further, the rare earths are a group of elements, and thus the tagged peptides do not normally share by the molecules that contain different elements. Liu et al (2006) developed a similar method called metal element chelate tag (MECT). As an inexpensive, well-known reagent used in the field of radiopharmacology, the diethylenetriamine pentaacetic acid bianhydride (DTPAA) is covalently coupled to primary amines of peptides, and the ligand is then chelated to the rare earth metals Y and Tb.…”

“…Peptides can be quantified by the relative signal intensities for the Y and Tb tagged pairs in ESI-MS. The method can offer convenient and rapid sample preparation and good sensitivity at the femtomole level (Liu et al, 2006).…”

Proteomics in China: Ready for prime time

“…In the area of identification of phosphopeptides by MS, a method of data processing for automatic verification of phosphorylated peptides was developed with the aid of multi-stage mass spectra [39]. In addition, many emphases have placed on large scale quantification of proteome for discovery of biological markers, drug targets and for tracing dynamic changes in life processes [40,41]. It is obvious that cutting-edge proteomic methods with high throughput, resolution, sensitivity and reproducibility and integrated ethnical systems are urgently demanded although substantial progress in proteomic techniques has been made.…”

Elemental labeling for the identification of proteinaceous-binding media in art works by ICP-MS

“…One of the first methods employed was a Cys‐specific labeling technique called element‐coded affinity tag based on tags containing different element‐coded metal chelates that incorporate rare earth elements into the macrocycle 1,4,7,10‐tetraazacyclododecane‐N,N′,N″,N‴‐tetraacetic acid (DOTA) . More recently, a new method using a metal element‐chelated tag (MECT) has been developed . In the MECT approach, to introduce the lanthanides the bicyclic anhydride of diethylenetriamine‐N,N,N′,N″,N‴ pentaacetic acid (DTPA) is used.…”

“…[24,25] More recently, a new method using a metal element-chelated tag (MECT) has been developed. [26][27][28] In the MECT approach, to introduce the lanthanides the bicyclic anhydride of diethylenetriamine-N,N,N′,N 00 ,N 000 pentaacetic acid (DTPA) is used. With respect to DOTA, this is a less expensive labeling agent that reacts with primary amines (amino terminus and internal Lys) present in the proteins.…”