Absolute SILAC for accurate quantitation of proteins in complex mixtures down to the attomole level

Mass spectrometry based proteomics can routinely identify hundreds of proteins in a single LC-MS run, and methods have been developed for relative quantitation between differentially treated samples using stable isotopes. However, absolute quantitation has so far required addition of a labeled standard late in the experimental workflow, introducing variability due to sample preparation. Here we present a new variant of the stable isotope labeling by amino acids in cell culture (SILAC) technique termed "Absolute SILAC" that allows accurate quantitation of selected proteins in complex mixtures. SILAC-labeled recombinant proteins produced in vivo or in vitro are used as internal standards, which are directly mixed into lysates of cells or tissues. This minimizes differences in sample processing between the isotope-labeled standard and its endogenous counterpart. We show that it is possible to quantify over several orders of magnitude, even in the background of a whole cell lysate. We furthermore devise a strategy to quantify peptides at or below their signal-to-noise level on hybrid ion trap instruments, shown here for the LTQ-Orbitrap. The data system triggers on peptides of the SILAC-labeled protein, initiating ion collection in a narrow mass range including the endogenous and labeled peptide. This strategy extends the regular detection limit of an LTQ-Orbitrap by at least an order of magnitude and accurately quantifies down to 150 attomole of protein in a cell lysate without any fractionation prior to LC-MS. We use Absolute SILAC to determine the copy number per cell of growth factor receptor-bound protein 2 (Grb2) in HeLa, HepG2, and C2C12 cells to 5.5 x 10(5), 8.8 x 10(5), and 5.7 x 10(5), respectively, in the exponential growth phase.     

Extending SILAC to proteomics of plant cell lines

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) is a widespread method for metabolic labeling of cells and tissues in quantitative proteomics; however, incomplete incorporation of the label has so far restricted its wider use in plants. Here, we argue that differential labeling by two different versions of the labeled amino acids renders SILAC fully applicable to dark-grown plant cell lines. By comparing Arabidopsis thaliana cell cultures labeled with two versions of heavy Lys (Lys-4 and Lys-8), we show that this simple modification of the SILAC protocol enables similar quantitation accuracy, precision, and reproducibility as conventional SILAC in animal cells.     

Study of neurotrophin-3 signaling in primary cultured neurons using multiplex stable isotope labeling with amino acids in cell culture

Conventional stable isotope labeling with amino acids in cell culture (SILAC) requires extensive metabolic labeling of proteins and therefore is difficult to apply to cells that do not divide or are unstable in SILAC culture. Using two different sets of heavy amino acids for labeling allows for straightforward SILAC quantitation using partially labeled cells because the two cell populations are always equally labeled. Here we report the application of this labeling strategy to primary cultured neurons. We demonstrated that protein quantitation was not compromised by incomplete labeling of the neuronal proteins. We used this method to study neurotrophin-3 (NT-3) signaling in primary cultured neurons. Surprisingly our results indicate TrkB signaling is a major component of the signaling network induced by NT-3 in cortical neurons. In addition, involvement of proteins such as VAMP2, Scamp1, and Scamp3 suggests that NT-3 may lead to enhanced exocytosis of synaptic vesicles.     

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.     

Relative protein quantification by isobaric SILAC with immonium ion splitting (ISIS)

Metabolic labeling techniques have recently become popular tools for the quantitative profiling of proteomes. Classical stable isotope labeling with amino acids in cell cultures (SILAC) uses pairs of heavy/light isotopic forms of amino acids to introduce predictable mass differences in protein samples to be compared. After proteolysis, pairs of cognate precursor peptides can be correlated, and their intensities can be used for mass spectrometry-based relative protein quantification. We present an alternative SILAC approach by which two cell cultures are grown in media containing isobaric forms of amino acids, labeled either with 13C on the carbonyl (C-1) carbon or 15N on backbone nitrogen. Labeled peptides from both samples have the same nominal mass and nearly identical MS/MS spectra but generate upon fragmentation distinct immonium ions separated by 1 amu. When labeled protein samples are mixed, the intensities of these immonium ions can be used for the relative quantification of the parent proteins. We validated the labeling of cellular proteins with valine, isoleucine, and leucine with coverage of 97% of all tryptic peptides. We improved the sensitivity for the detection of the quantification ions on a pulsing instrument by using a specific fast scan event. The analysis of a protein mixture with a known heavy/light ratio showed reliable quantification. Finally the application of the technique to the analysis of two melanoma cell lines yielded quantitative data consistent with those obtained by a classical two-dimensional DIGE analysis of the same samples. Our method combines the features of the SILAC technique with the advantages of isobaric labeling schemes like iTRAQ. We discuss advantages and disadvantages of isobaric SILAC with immonium ion splitting as well as possible ways to improve it.     

Preventing arginine-to-proline conversion in a cell-line-independent manner during cell cultivation under stable isotope labeling by amino acids in cell culture (SILAC) conditions

Quantitative proteomics has increasingly gained impact in life science research as a tool to describe changes in protein expression between different cellular states. Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful technique for relative quantification of proteins. However, the accuracy of quantification is impaired by the metabolic conversion of arginine to proline resulting in additional heavy labeled proline peptide satellites. Here we reinvestigated the addition of unlabeled proline during cell cultivation under SILAC conditions considering several thousand peptides and demonstrated that the arginine-to-proline conversion is prevented independent of the cell line used.     

Use of stable isotope labeling by amino acids in cell culture as a spike-in standard in quantitative proteomics

Mass spectrometry (MS)-based proteomics is increasingly applied in a quantitative format, often based on labeling of samples with stable isotopes that are introduced chemically or metabolically. In the stable isotope labeling by amino acids in cell culture (SILAC) method, two cell populations are cultured in the presence of heavy or light amino acids (typically lysine and/or arginine), one of them is subjected to a perturbation, and then both are combined and processed together. In this study, we describe a different approach--the use of SILAC as an internal or 'spike-in' standard--wherein SILAC is only used to produce heavy labeled reference proteins or proteomes. These are added to the proteomes under investigation after cell lysis and before protein digestion. The actual experiment is therefore completely decoupled from the labeling procedure. Spike-in SILAC is very economical, robust and in principle applicable to all cell- or tissue-based proteomic analyses. Applications range from absolute quantification of single proteins to the quantification of whole proteomes. Spike-in SILAC is especially advantageous when analyzing the proteomes of whole tissues or organisms. The protocol describes the quantitative analysis of a tissue sample relative to super-SILAC spike-in, a mixture of five SILAC-labeled cell lines that accurately represents the tissue. It includes the selection and preparation of the spike-in SILAC standard, the sample preparation procedure, and analysis and evaluation of the results.     

用SILAC技术研究感染H5N1禽流感病毒后A549肺癌细胞蛋白质组的表达变化

目的：鉴定高致病性H5N1禽流感病毒感染A549肺癌细胞后,细胞蛋白质组的表达变化,并鉴定特异分子通路的改变及其涉及的关键蛋白质分子。方法：利用稳定同位素标记氨基酸技术（SILAC）标记A549细胞,得到“重标”或“轻标”的A549细胞;“重标”细胞感染高致病性F15N1禽流感病毒24h后提取细胞总蛋白,与从未感染病毒的“轻标”细胞中提取的总蛋白等量混合,酶解肽段,经正交反相色谱分离后用质谱鉴定,对数据进行定性和定量分析。结果：共鉴定到3504个蛋白质,有定量信息的蛋白质为2469个,病毒感染后表达量升高的蛋白质为72个,表达量降低的蛋白质为66个,其中包括参与多个分子调控途径如RNA剪接体、干扰素诱导通路、泛素化通路、胰岛素通路等的蛋白质。结论：建立了利用SILAC技术研究宿主细胞一病毒相互作用的方法,发现了高致病性H5N1禽流感病毒感染宿主细胞相关的关键蛋白质,为探索H5N1病毒致病的分子机理提供了理论基础。     

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.     

Quantitative Proteomics Employing Primary Amine Affinity Tags

A proteomics-based method using stable isotope labeling to assess the relative abundance of peptides or proteins is described. Bradykinin and carbonic anhydrase were labeled with sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate, a membrane impermeant reagent that is reactive with primary amines. Specificity of the label to primary amines was demonstrated using tandem mass spectrometry. Also, relative quantitation was achieved by secondary labeling with natural isotopic abundance and stable isotope-labeled methyl iodide. We believe this to be an effective stable isotope-labeling method for quantitative proteomics.     

Characterization of a SILAC method for proteomic analysis of primary rat microglia

Microglia play important and dynamic roles in mediating a variety of physiological and pathological processes during the development, normal function and degeneration of the central nervous system. Application of SILAC‐based proteomic analysis would greatly facilitate the identification of cellular pathways regulating the multifaceted phenotypes of microglia. We and others have successfully SILAC‐labeled immortalized murine microglial cell lines in previous studies. In this study, we report the development and evaluation of a SILAC‐labeled primary rat microglia model. Although the isotope labeling scheme for primary microglia is drastically different from that of immortalized cell lines, our de novo and uninterrupted primary culture labeling protocol (DUP‐SILAC) resulted in sufficient incorporation of SILAC labels for mass spectrometry‐based proteomic profiling. In addition, label incorporation did not alter their morphology and response to endotoxin stimulation. Proteomic analysis of the endotoxin‐stimulated SILAC‐labeled primary microglia identified expected as well as potentially novel activation markers and pro‐inflammatory pathways that could be quantified in a more physiologically relevant cellular model system compared to immortalized cell lines. The establishment of primary microglia SILAC model will further expand our capacity for global scale proteomic profiling of pathways under various physiological and pathological conditions. Proteomic MS data are available via ProteomeXchange with identifier PXD002759.     

Protein labeling by iTRAQ: a new tool for quantitative mass spectrometry in proteome research

A novel, MS-based approach for the relative quantification of proteins, relying on the derivatization of primary amino groups in intact proteins using isobaric tag for relative and absolute quantitation (iTRAQ) is presented. Due to the isobaric mass design of the iTRAQ reagents, differentially labeled proteins do not differ in mass; accordingly, their corresponding proteolytic peptides appear as single peaks in MS scans. Because quantitative information is provided by isotope-encoded reporter ions that can only be observed in MS/MS spectra, we analyzed the fragmentation behavior of ESI and MALDI ions of peptides generated from iTRAQ-labeled proteins using a TOF/TOF and/or a QTOF instrument. We observed efficient liberation of reporter ions for singly protonated peptides at low-energy collision conditions. In contrast, increased collision energies were required to liberate the iTRAQ label from lysine side chains of doubly charged peptides and, thus, to observe reporter ions suitable for relative quantification of proteins with high accuracy. We then developed a quantitative strategy that comprises labeling of intact proteins by iTRAQ followed by gel electrophoresis and peptide MS/MS analyses. As proof of principle, mixtures of five different proteins in various concentration ratios were quantified, demonstrating the general applicability of the approach presented here to quantitative MS-based proteomics.     

Selective detection of membrane proteins without antibodies: a mass spectrometric version of the Western blot

A method has been developed, called the mass western experiment in analogy to the Western blot, to detect the presence of specific proteins in complex mixtures without the need for antibodies. Proteins are identified with high sensitivity and selectivity, and their abundances are compared between samples. Membrane protein extracts were labeled with custom isotope-coded affinity tag reagents and digested, and the labeled peptides were analyzed by liquid chromatography-tandem mass spectrometry. Ions corresponding to anticipated tryptic peptides from the proteins of interest were continuously subjected to collision-induced dissociation in an ion trap mass spectrometer; heavy and light isotope-coded affinity tag-labeled peptides were simultaneously trapped and fragmented accomplishing identification and quantitation in a single mass spectrum. This application of ion trap selective reaction monitoring maximizes sensitivity, enabling analysis of peptides that would otherwise go undetected. The cell surface proteins prostate stem cell antigen (PSCA) and ErbB2 were detected in prostate and breast tumor cell lines in which they are expressed in known abundances spanning orders of magnitude.     

Detection of in situ labeled cell surface proteins by mass spectrometry: application to the membrane subproteome of human mammary epithelial cells

Characterization of the surface exposed membrane subproteome of human mammary epithelial cells (strain 184 A1L5) implemented lysine specific in situ labeling of the proteins using sulfosuccinimidyl-6-(biotinamido)hexanoate, followed by enrichment of the biotinylated, tryptically digested peptides, and then liquid chromatography-tandem mass spectrometry analysis of the labeled peptides. Probing the membrane subproteome in this manner yielded unambiguous identification of proteins situated on the cell surface. The method reported can be adapted to include stable isotope labeling of proteins for quantitation of changes occurring on the cell surface in response to specific perturbations.     

Identifying and quantifying in vivo methylation sites by heavy methyl SILAC

Protein methylation is a stable post-translational modification (PTM) with important biological functions. It occurs predominantly on arginine and lysine residues with varying numbers of methyl groups, such as mono-, di- or trimethyl lysine. Existing methods for identifying methylation sites are laborious, require large amounts of sample and cannot be applied to complex mixtures. We have previously described stable isotope labeling by amino acids in cell culture (SILAC) for quantitative comparison of proteomes. In heavy methyl SILAC, cells metabolically convert [(13)CD(3)]methionine to the sole biological methyl donor, [(13)CD(3)]S-adenosyl methionine. Heavy methyl groups are fully incorporated into in vivo methylation sites, directly labeling the PTM. This provides markedly increased confidence in identification and relative quantitation of protein methylation by mass spectrometry. Using antibodies targeted to methylated residues and analysis by liquid chromatography-tandem mass spectrometry, we identified 59 methylation sites, including previously unknown sites, considerably extending the number of in vivo methylation sites described in the literature.     

SILAC zebrafish for quantitative analysis of protein turnover and tissue regeneration

Defective tissue regeneration is thought to contribute to several human diseases, including neurodegenerative disorders, heart failure and various lung diseases. Boosting the regenerative capacity has been suggested a possible therapeutic approach. Methods to metabolically label newly synthesized proteins in vivo with stable isotopic forms of amino acids holds promise for the study of protein turnover and tissue regeneration that are fundamental to the sustained life of all organisms. Here, we used the "stable isotope labeling with amino acids in cell culture" (SILAC) approach to explore normal protein turnover and tissue regeneration in adult zebrafish. The ratio of labeled and unlabeled proteins/peptides in specific organs of zebrafish fed a SILAC diet containing (13)C(6)-labeled lysine was determined by liquid chromatography and tandem mass spectrometry. Labeling was highest in tissues with high regenerative capacity, including intestine, liver, and fin, whereas brain and heart displayed the lowest labeling. Proteins with high degree of labeling were mainly involved in catalytic or transport activity pathways. The technique also verified increased protein synthesis during regeneration of the caudal fin following amputation. This newly developed SILAC zebrafish model constitutes a novel tool to analyze tissue regeneration in an animal model amenable to genetic and pharmacologic manipulation.