Stable isotope labeling approaches

Stable isotope labeling entails the incorporation of stable heavy atoms such as ¹³C and ¹⁵N into specific biomolecular entities, as previously reviewed[24]. In most cases, these labels are chemically or metabolically introduced into peptides or proteins. One of the first chemical labeling strategies adopted for protein quantification by mass spectrometry was the isotope-coded affinity tag (ICAT) technique[25]. In this approach, the sulfhydryl groups in cysteine residues are covalently modified by the ICAT reagents containing “light” or “heavy” isotopes. The presence of the light or heavy ICAT tags leads to the separation and concomitant quantification of modified peptides during the precursor ion measurements in the first mass spectrometry process (MS1).

Recently, chemical labeling strategies utilizing isobaric tags, i.e., tags with the same molecular weight, developed for relative and absolute quantification (iTRAQ)[26] and tandem mass tags (TMT)[27] have gained popularity in proteomics. iTRAQ and TMT reagents differ from ICAT reagents in that the ε-amino group of lysine and α-amino group of the N-terminal residue in peptides are modified. The labeled peptides are quantified during the second mass spectrometry process (MS2) when the tags are released upon fragmentation of the peptides (Figure ​(Figure1).1). Advantages of the isobaric tags include a multiplexing capacity of up to eight separate samples in a single mass spectrometric run. Additionally, because isobaric tags modify amino groups, which are more abundant than sulfhydryl groups in most proteins, the coverage of quantification by iTRAQ and TMT is also greater than by ICAT.

Schematic overview of labeling strategies used in quantitative proteomics. Chemical labeling utilizes the isobaric tags for relative and absolute quantification (iTRAQ) and the tandem mass tags (TMT). In this approach, proteolytic peptides from separate samples are labeled with discrete isobaric tags and pooled. Precursor peptide ions are fragmented (MS2) to generate reporter ions with distinct m/z, whose relative intensities represent the relative abundances of the peptides producing the corresponding reporter ion. Metabolic labeling represented by the stable isotope labeling with amino acids in cell culture (SILAC) strategy takes advantage of the metabolic incorporation of heavy amino acids into mature proteins. In this strategy, the relative peak intensities (MS1) represent the abundances of the precursor peptide ions.

A large number of studies quantifying the proteomes of gastric cancer using the iTRAQ approach have been reported. Morisaki et al[28] applied iTRAQ to identify potential biomarkers in gastric cancer stem cells and identified nine proteins that were overproduced in gastric cancer stem cells. Using iTRAQ, Subbannayya et al[29] defined a set of potential biomarkers in sera from gastric cancer patients. In their study, more than 50 proteins were found to exhibit altered levels in samples from gastric cancer patients.

TMT has also been used to quantify gastric cancer proteomes. Gao et al[30] found that 234 mitochondrial protein genes were differentially expressed in gastric cancer using TMT. In another study employing TMT, Gao et al[31] revealed that 82 plasma membrane proteins were dysregulated in gastric cancer.

An alternative to stable isotope labeling technique is metabolic labeling. This approach takes advantage of the metabolic incorporation of heavy isotopes in live cells under culture conditions. Quantification by metabolic labeling is less error-prone than chemical labeling because the labels are introduced before the samples are prepared. Stable isotope labeling with amino acids in cell culture (SILAC)[32] is one of the most popular metabolic labeling techniques (Figure ​(Figure1).1). Developed by Ong et al[32], SILAC labels proteins in the cells by growing them in the medium containing heavy amino acids. The most common heavy amino acids used in SILAC are lysine-4, lysine-8, arginine-6, and arginine-10. Different combinations of heavy lysines and arginines can be used such that up to three simultaneous quantifications are possible as follows: a light sample (Lys-0 and Arg-0), medium sample (Lys-4 and Arg-6), and heavy sample (Lys-8 and Arg-10).

As trypsin is the most popular protease used for the preparation of peptide mixtures, which cleaves the carboxyl side of lysine or arginine, the use of heavy lysine and arginine in SILAC helps increase the coverage of quantification by ensuring that every peptide analyzed by the mass spectrometer contains at least one heavy amino acid. Like labeling with ICAT, the quantification of proteins labeled with the SILAC approach is carried out by comparing the intensities of precursor peptide ions in the MS1 process. Quantification employing the SILAC method has been applied in gastric cancer proteomics. Marimuthu et al[33] studied the secretomes from neoplastic and non-neoplastic gastric epithelial cells using SILAC. The authors identified 263 proteins that were upregulated in gastric cancer-derived cells compared to non-neoplastic gastric epithelial cells.