Shao En Ong

Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics

Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures. Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins. Mammalian cell lines are grown in media lacking a standard essential amino acid but supplemented with a non-radioactive, isotopically labeled form of that amino acid, in this case deuterated leucine (Leu-d3). We find that growth of cells maintained in these media is no different from growth in normal media as evidenced by cell morphology, doubling time, and ability to differentiate. Complete incorporation of Leu-d3 occurred after five doublings in the cell lines and proteins studied. Protein populations from experimental and control samples are mixed directly after harvesting, and mass spectrometric identification is straightforward as every leucine-containing peptide incorporates either all normal leucine or all Leu-d3. We have applied this technique to the relative quantitation of changes in protein expression during the process of muscle cell differentiation. Proteins that were found to be up-regulated during this process include glyceraldehyde-3-phosphate dehydrogenase, fibronectin, and pyruvate kinase M2. SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system.

Nucleolar proteome dynamics

The nucleolus is a key organelle that coordinates the synthesis and assembly of ribosomal subunits and forms in the nucleus around the repeated ribosomal gene clusters. Because the production of ribosomes is a major metabolic activity, the function of the nucleolus is tightly linked to cell growth and proliferation, and recent data suggest that the nucleolus also plays an important role in cell-cycle regulation, senescence and stress responses. Here, using mass-spectrometry-based organellar proteomics and stable isotope labelling, we perform a quantitative analysis of the proteome of human nucleoli. In vivo fluorescent imaging techniques are directly compared to endogenous protein changes measured by proteomics. We characterize the flux of 489 endogenous nucleolar proteins in response to three different metabolic inhibitors that each affect nucleolar morphology. Proteins that are stably associated, such as RNA polymerase I subunits and small nuclear ribonucleoprotein particle complexes, exit from or accumulate in the nucleolus with similar kinetics, whereas protein components of the large and small ribosomal subunits leave the nucleolus with markedly different kinetics. The data establish a quantitative proteomic approach for the temporal characterization of protein flux through cellular organelles and demonstrate that the nucleolar proteome changes significantly over time in response to changes in cellular growth conditions.

Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome

In signal transduction in eukaryotes, protein phosphorylation is a key event. To understand signaling processes, we must first acquire an inventory of phosphoproteins and their phosphorylation sites under different conditions. Because phosphorylation is a dynamic process, elucidation of signaling networks also requires quantitation of these phosphorylation events. In this article, we outline several methods for enrichment of phosphorylated proteins and peptides and discuss various options for their identification and quantitation with special emphasis on mass spectrometry-based techniques.

Trypsin Cleaves Exclusively C-terminal to Arginine and Lysine Residues

Almost all large-scale projects in mass spectrometry-based proteomics use trypsin to convert protein mixtures into more readily analyzable peptide populations. When searching peptide fragmentation spectra against sequence databases, potentially matching peptide sequences can be required to conform to tryptic specificity, namely, cleavage exclusively C-terminal to arginine or lysine. In many published reports, however, significant numbers of proteins are identified by non-tryptic peptides. Here we use the sub-parts per million mass accuracy of a new ion trap Fourier transform mass spectrometer to achieve more than a 100-fold increased confidence in peptide identification compared with typical ion trap experiments and show that trypsin cleaves solely C-terminal to arginine and lysine. We find that non-tryptic peptides occur only as the C-terminal peptides of proteins and as breakup products of fully tryptic peptides N-terminal to an internal proline. Simulating lower mass accuracy led to a large number of proteins erroneously identified with non-tryptic peptide hits. Our results indicate that such peptide hits in previous studies should be re-examined and that peptide identification should be based on strict trypsin specificity.

A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling.

Mass spectrometry–based proteomics can reveal protein-protein interactions on a large scale, but it has been difficult to separate background binding from functionally important interactions and still preserve weak binders. To investigate the epidermal growth factor receptor (EGFR) pathway, we employ stable isotopic amino acids in cell culture (SILAC) to differentially label proteins in EGF-stimulated versus unstimulated cells. Combined cell lysates were affinity-purified over the SH2 domain of the adapter protein Grb2 (GST-SH2 fusion protein) that specifically binds phosphorylated EGFR and Src homologous and collagen (Shc) protein. We identified 228 proteins, of which 28 were selectively enriched upon stimulation. EGFR and Shc, which interact directly with the bait, had large differential ratios. Many signaling molecules specifically formed complexes with the activated EGFR-Shc, as did plectin, epiplakin, cytokeratin networks, histone H3, the glycosylphosphatidylinositol (GPI)-anchored molecule CD59, and two novel proteins. SILAC combined with modification-based affinity purification is a useful approach to detect specific and functional protein-protein interactions.

A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC)

Stable isotope labeling by amino acids in cell culture (SILAC) is a simple, robust, yet powerful approach in mass spectrometry (MS)-based quantitative proteomics. SILAC labels cellular proteomes through normal metabolic processes, incorporating non-radioactive, stable isotope-containing amino acids in newly synthesized proteins. Growth medium is prepared where natural (“light”) amino acids are replaced by “heavy” SILAC amino acids. Cells grown in this medium incorporate the heavy amino acids after five cell doublings and SILAC amino acids have no effect on cell morphology or growth rates. When light and heavy cell populations are mixed, they remain distinguishable by MS, and protein abundances are determined from the relative MS signal intensities. SILAC provides accurate relative quantification without any chemical derivatization or manipulation and enables development of elegant functional assays in proteomics. In this protocol, we describe how to apply SILAC and the use of nano-scale liquid chromatography coupled to electrospray ionization mass spectrometry for protein identification and quantification. This procedure can be completed in 8 days.

Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics

To study the global dynamics of phosphotyrosine-based signaling events in early growth factor stimulation, we developed a mass spectrometric method that converts temporal changes to differences in peptide isotopic abundance. The proteomes of three cell populations were metabolically encoded with different stable isotopic forms of arginine. Each population was stimulated by epidermal growth factor for a different length of time, and tyrosine-phosphorylated proteins and closely associated binders were affinity purified. Arginine-containing peptides occurred in three forms, which were quantified; we then combined two experiments to generate five-point dynamic profiles. We identified 81 signaling proteins, including virtually all known epidermal growth factor receptor substrates, 31 novel effectors and the time course of their activation upon epidermal growth factor stimulation. Global activation profiles provide an informative perspective on cell signaling and will be crucial to modeling signaling networks in a systems biology approach.

Mass spectrometric-based approaches in quantitative proteomics

Classically, experiments aimed at studying changes in protein expression have always followed a small set of proteins. This focused approach was necessary since tools to efficiently analyze large numbers of proteins were simply not available. Large-scale quantitative proteomics promises to produce reams of data that previously would have taken decades to measure with classical methods. Mass spectrometry is already a well-established protein identification tool and recent methodological developments indicate that it can also be successfully applied to extract quantitative data of protein abundance. From the first reports 4 years ago, numerous schemes to take advantage of stable isotope nuclei incorporation in proteins and peptides have been developed. Here we review the benefits and pitfalls of some of the most commonly used protocols, focusing on a procedure now being used extensively in our laboratory, stable isotope labeling with amino acids in cell culture (SILAC). The basic theory, application, and data analysis of a SILAC experiment are discussed. The emerging nature of these techniques and the rapid pace of technological development make forecasting the directions of the field difficult but we speculate that SILAC will soon be a key tool of quantitative proteomics.

An evaluation of the use of two-dimensional gel electrophoresis in proteomics

With whole genomes being sequenced almost routinely, the next logical step towards a better understanding of cellular mechanisms lies in studying the functional units of gene expression-proteins. One of the fundamental approaches in proteomics is the use of two-dimensional gel electrophoresis as a mode of separation and visualization of complex protein mixtures. Despite several limitations of the method, its ability to separate large numbers of proteins, including their post-translationally modified forms, ensures that it will continue to be popular in several well-defined areas of proteomics. In this article, we discuss the merits and drawbacks of two-dimensional gels and compare them with alternative systems such as one-dimensional gels and liquid chromatography-based separation methods. In the wake of recent advances in mass spectrometry and related areas, we outline areas where two-dimensional gels can best be utilized as the preferred separation method in proteomic strategies.

iTRAQ Labeling is Superior to mTRAQ for Quantitative Global Proteomics and Phosphoproteomics

Labeling of primary amines on peptides with reagents containing stable isotopes is a commonly used technique in quantitative mass spectrometry. Isobaric labeling techniques such as iTRAQ™ or TMT™ allow for relative quantification of peptides based on ratios of reporter ions in the low m/z region of spectra produced by precursor ion fragmentation. In contrast, nonisobaric labeling with mTRAQ™ yields precursors with different masses that can be directly quantified in MS1 spectra. In this study, we compare iTRAQ- and mTRAQ-based quantification of peptides and phosphopeptides derived from EGF-stimulated HeLa cells. Both labels have identical chemical structures, therefore precursor ion- and fragment ion-based quantification can be directly compared. Our results indicate that iTRAQ labeling has an additive effect on precursor intensities, whereas mTRAQ labeling leads to more redundant MS2 scanning events caused by triggering on the same peptide with different mTRAQ labels. We found that iTRAQ labeling quantified nearly threefold more phosphopeptides (12,129 versus 4,448) and nearly twofold more proteins (2,699 versus 1,597) than mTRAQ labeling. Although most key proteins in the EGFR signaling network were quantified with both techniques, iTRAQ labeling allowed quantification of twice as many kinases. Accuracy of reporter ion quantification by iTRAQ is adversely affected by peptides that are cofragmented in the same precursor isolation window, dampening observed ratios toward unity. However, because of tighter overall iTRAQ ratio distributions, the percentage of statistically significantly regulated phosphopeptides and proteins detected by iTRAQ and mTRAQ was similar. We observed a linear correlation of logarithmic iTRAQ to mTRAQ ratios over two orders of magnitude, indicating a possibility to correct iTRAQ ratios by an average compression factor. Spike-in experiments using peptides of defined ratios in a background of nonregulated peptides show that iTRAQ quantification is less accurate but not as variable as mTRAQ quantification.