Quantitative proteomics for cancer biomarker discovery

The mass spectrometry (MS)-based quantitative proteomics is powerful to discover disease biomarkers that can provide diagnostic, prognostic and therapeutic targets, and it also can address important problems in clinical and translational medical research. The current status of MS-based quantification strategy and technical advances of several main quantitative assays (two-dimensional (2-D) gel-based methods, stable isotope labeling with amino acids in cell culture (SILAC), isotope-coded affinity tag (ICAT), the isobaric tags for relative and absolute quantification (iTRAQ), 1?O labeling, absolute quantitation and label-free quantitation) have been summarized and reviewed. At present, except 2-D gel-based methods, several stable isotope labeling quantitative techniques, including SILAC, ICAT and iTRAQ, etc, have been widely applied in identification of differential expression of proteins, post-translational modifications and protein-protein interactions in order to look for novel candidate cancer biomarkers from different physiological states of cells, body fluids or tissue samples. Also, the advantages and challenges of different quantitative proteomic approaches are discussed in identification and validation of candidate targets.     

A new protocol of analyzing isotope-coded affinity tag data from high-resolution LC-MS spectrometry

Isotope-coded affinity tags (ICAT) is a labeling technique that provides insights into quantitative molecular changes. In this paper, we propose a new protocol to identify and analyze ICAT labeled peak pairs in high-resolution LC-MS data. Our major contributions are: (1) we use isotope distance constraint, ICAT distance constraint, and LC-span constraint to identify ICAT labeled peak pairs and (2) we propose to trigger tandem MS/MS scanning based on the ratio estimation value of identified ICAT peak pairs instead of the peak intensity values. Compared with current approaches that choose peaks with high intensity values for tandem MS/MS scanning, the new protocol can improve the scanning efficiency and accuracy.     

Identification of redox sensitive thiols of protein disulfide isomerase using isotope coded affinity technology and mass spectrometry

Regulation of the redox state of protein disulfide isomerase (PDI) is critical for its various catalytic functions. Here we describe a procedure utilizing isotope-coded affinity tag (ICAT) technology and mass spectrometry that quantitates relative changes in the dynamic thiol and disulfide states of human PDI. Human PDI contains six cysteine residues, four present in two active sites within the a and a' domains, and two present in the b' domain. ICAT labeling of human PDI indicates a difference between the redox state of the two active sites. Furthermore, under auto-oxidation conditions an approximately 80% decrease in available thiols within the a domain was detected. Surprisingly, the redox state of one of the two cysteines, Cys-295, within the b' domain was altered between the fully reduced and the auto-oxidized state of PDI while the other b' domain cysteine remained fully reduced. An interesting mono- and dioxidation modification of an invariable tryptophan residue, Trp-35, within the active site was also mapped by tandem mass spectrometry. Our findings indicate that ICAT methodology in conjunction with mass spectrometry represents a powerful tool to monitor changes in the redox state of individual cysteine residues within PDI under various conditions.     

Proteomic analysis of Pseudomonas aeruginosa grown under magnesium limitation

In this study, large-scale qualitative and quantitative proteomic technology was applied to the analysis of the opportunistic bacterial pathogen Pseudomonas aeruginosa grown under magnesium limitation, an environmental condition previously shown to induce expression of various virulence factors. For quantitative analysis, whole cell and membrane proteins were differentially labeled with isotope-coded affinity tag (ICAT) reagents and ICAT reagent-labeled peptides were separated by two-dimensional chromatography prior to analysis by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) in an ion trap mass spectrometer (ITMS). To increase the number of protein identifications, gas-phase fractionation (GPF) in the m/z dimension was employed for analysis of ICAT peptides derived from whole cell extracts. The experiments confirmed expression of 1331 P. aeruginosa proteins of which 145 were differentially expressed upon limitation of magnesium. A number of conserved Gram-negative magnesium stress-response proteins involved in bacterial virulence were among the most abundant proteins induced in low magnesium. Comparative ICAT analysis of membrane versus whole cell protein indicated that growth of P. aeruginosa in low magnesium resulted in altered subcellular compartmentalization of large enzyme complexes such as ribosomes. This result was confirmed by 2-D PAGE analysis of P. aeruginosa outer membrane proteins. This study shows that large-scale quantitative proteomic technology can be successfully applied to the analysis of whole bacteria and to the discovery of functionally relevant biologic phenotypes.     

Relative quantitation of proteins fractionated by the ProteomeLab™ PF 2D system using isobaric tags for relative and absolute quantitation (iTRAQ)

We describe an optimised protocol for application of isobaric tags for relative and absolute quantitation (iTRAQ) and tandem mass spectrometry to obtain relative quantitative data from peptides derived from tryptic digestions of proteins fractionated by using the 2D liquid-phase ProteomeLab™ PF 2D technique. This methodology is suitable for the quantitation of proteins from a pool of co-eluting proteins which are often difficult to identify for the purpose of candidate protein selection for biologically relevant qualitative/quantitative changes under experimental conditions or in disease states. iTRAQ quantitation also facilitates the possibility of result to result comparison using other methodologies such as UV protein quantitation via the ProteomeLab™ PF 2D technique. The optimised protocol outlined here allows relative quantitation by MALDI-TOF/TOF mass spectrometry with high sensitivity and without the need to perform 2D HPLC separation of labelled peptides. The overall outcome is the simplification in the data complexity and the ease of use of the labelling protocol. This study is dedicated to Dr. Josef Chmelik in memory of his contribution and constant inspiration.     

Review of molecular modification techniques for improved detection of biomolecules by mass spectrometry

After a soft ionizing method was established, MS (mass spectrometry) has become a more common tool in biochemistry because soft ionization made it possible to detect large molecules such as proteins. Many kinds of applications were established to further utilize MS for the identification or quantitation of biomolecules. In this review, we introduce recent applications with special focus on chemical modification techniques and chemical probes developed for the MS determination of biomolecules.     

亲和分离在蛋白质泛素化修饰研究中的应用进展

蛋白质泛素化是真核生物最普遍、最复杂的翻译后修饰方式之一,在细胞的信号转导、生长、发育、代谢等生命过程中发挥着重要作用。泛素化过程的失调则与神经退行性疾病、炎症反应、癌症等重大疾病的发生发展密切相关。分析和研究蛋白质泛素化的结构与功能,可望为认识生命、探索疾病调控内在规律和发现新的诊断策略提供重要信息。生命体系的高度复杂性,泛素化修饰位点、结构类型的多变和多样性,时空动态变化等特点给蛋白质泛素化分析研究带来了巨大的挑战。亲和分离以其高选择性成为泛素化蛋白质结构与功能研究的有力工具。免疫亲和分离法基于抗原-抗体相互作用,是最为经典的分离分析方法,已广泛应用于泛素化蛋白质或肽段的富集分离。源于天然泛素受体的泛素结合结构域(ubiquitin binding domains, UBDs)可与泛素或多聚泛素链相互作用。UBDs和基于此发展起来的串联泛素结合实体(tandem ubiquitin-binding entities, TUBEs)已成为蛋白质泛素化功能研究的热门识别分子。各种多肽类化合物的发展也为蛋白质泛素化的结构和功能解析提供新工具。此外,多种亲和识别配基的联合使用,在蛋白质泛素化修饰的高特异性、高灵敏度分析中展现了独特的优势,为认识生命体内的泛素化修饰提供了重要保障。该文对亲和分离方法在蛋白质泛素化修饰分析中的应用及进展进行了综述。     

Quantitative Fourier transform ion cyclotron resonance mass spectrometry--the determination of creatinine by isotope dilution mass spectrometry

Accurate quantitation has been demonstrated on many different types of mass spectrometer. However, quantitative applications of Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) have been limited. In this study, the quantitative potential of FTICRMS has been investigated using an exact matching isotope dilution method for the determination of creatinine in serum. Creatinine is an important clinical biomarker and its measurement is used as an assessment of renal function. The quantitation of creatinine was selected because a high-accuracy high-performance liquid chromatography/mass spectrometry (HPLC/MS) determination using a triple quadrupole mass spectrometer has already been successfully developed in-house. Therefore, a direct comparison of the quantitative capability of FTICRMS could be made against an established method. The accuracy of the quantitation of creatinine was found to be equivalent to that obtained using LC/MS. However, the expanded measurement uncertainty (k = 2) was larger, at 6%, when using FTICRMS compared with 1% when using HPLC/MS with the triple quadrupole mass spectrometer.     

"Mass defect" tags for biomolecular mass spectrometry

We present a new class of "mass defect" tags with utility in biomolecular mass spectrometry. These tags, incorporating element(s) with atomic numbers between 17 (Cl) and 77 (Ir), have a substantially different nuclear binding energy (mass defect) from the elements common to biomolecules. This mass defect yields a readily resolvable mass difference between tagged and untagged species in high-resolution mass spectrometers. We present the use of a subset of these tags in a new protein sequencing application. This sequencing technique has advantages over existing mass spectral protein identification methodologies: intact proteins are quickly sequenced and unambiguously identified using only an inexpensive, robust mass spectrometer. We discuss the potential broader utility of these tags for the sequencing of other biomolecules, differential display applications and combinatorial methods.     

金属标记结合高效液相色谱-选择离子监测质谱的蛋白质绝对定量新方法

建立了金属标记结合高效液相色谱-选择性离子监测质谱(SIM)的蛋白质绝对定量新方法。实验考察了金属标记效率、金属标记的稳定性、标记后肽段的色谱保留和质谱行为、新定量方法的线性范围和准确度。实验结果表明金属标记具有标记效率高,稳定性好,色谱保留行为一致等优点。另外,金属标记-选择离子监测质谱绝对定量方法灵敏度高,其定量限低至1 fmol,线性范围为1~500 fmol,线性范围内R2值大于0.99,具有良好的线性关系;经过测量,标准肽段的回收率为117.01%,说明该方法具有较高的准确度。将该方法应用于腾冲嗜热菌中烯醇酶蛋白的定量分析,相对标准偏差为5.47%,表明该方法的精密度高。以上结果表明该方法可以用于生物样本中的蛋白质的绝对定量分析,为比较简单的生物样本中蛋白质的绝对定量方法提供了一种新的选择。     

Comparative analysis of cleavable azobenzene-based affinity tags for bioorthogonal chemical proteomics

The advances in bioorthogonal ligation methods have provided new opportunities for proteomic analysis of newly synthesized proteins, posttranslational modifications, and specific enzyme families using azide/alkyne-functionalized chemical reporters and activity-based probes. Efficient enrichment and elution of azide/alkyne-labeled proteins with selectively cleavable affinity tags are essential for protein identification and quantification applications. Here, we report the synthesis and comparative analysis of Na?S?O?-cleavable azobenzene-based affinity tags for bioorthogonal chemical proteomics. We demonstrated that ortho-hydroxyl substituent is required for efficient azobenzene-bond cleavage and show that these cleavable affinity tags can be used to identify newly synthesized proteins in bacteria targeted by amino acid chemical reporters as well as their sites of modification on endogenously expressed proteins. The azobenzene-based affinity tags are compatible with in-gel, in-solution, and on-bead enrichment strategies and should afford useful tools for diverse bioorthogonal proteomic applications.     

Quantitative determination of noncovalently bound acridinium in protein conjugates by liquid chromatography/electrospray ion trap mass spectrometry

A sensitive and robust liquid chromatography/electrospray ion trap mass spectrometry (LC/MS/MS) method has been developed for the quantitative determination of noncovalently bound acridinium free acid in protein-acridinium conjugates. The lower level of quantitation (LOQ) for acridinium free acid was determined to be 0.6 ng. The assay was validated with a linear concentration range of 0.6-60 ng. The method requires minimum sample handling and is specific, reproducible, and provides a new aspect for protein-acridinium conjugate characterization.     

Clickable report tags for identification of modified peptides by mass spectrometry

The identification and quantification of modified peptides are critical for the functional characterization of post-translational protein modifications (PTMs) to elucidate their biological function. Nowadays, quantitative mass spectrometry coupled with various bioinformatic pipelines has been successfully used for the determination of a wide range of PTMs. However, direct characterization of low abundant protein PTMs in bottom-up proteomic workflow remains challenging. Here, we present the synthesis and evaluation of tandem mass spectrometry tags (TMT) which are introduced via click-chemistry into peptides bearing alkyne handles. The fragmentation properties of the two mass tags were validated and used for screening in a model system and analysis of AMPylated proteins. The presented tags provide a valuable tool for diagnostic peak generation to increase confidence in the identification of modified peptides and potentially for direct peptide-PTM quantification from various experimental conditions.     

Proteome analysis of camptothecin-treated cortical neurons using isotope-coded affinity tags

Isotope-coded affinity tags (ICATs) were employed to identify and quantitate changes in protein expression between control and camptothecin-treated mouse cortical neurons. Proteins extracted from control cortical neurons and those treated with camptothecin were labeled with the light and heavy isotopic versions of the ICAT reagents, respectively. ICAT-labeled samples were combined, proteolytically digested, and the derivatized peptides isolated using immobilized avidin chromatography. The peptides thus isolated were analyzed by reversed-phase liquid chromatography coupled directly to either a conventional ion-trap mass spectrometer (IT-MS) or a Fourier transform ion cyclotron resonance mass spectrometer (FTICR). While a majority of the peptide identifications were accomplished using IT-MS, FTICR was used to quantitate the relative abundances of the ICAT-labeled peptides taking advantage of its high resolution, sensitivity, and duty cycle. By using this combination of MS technologies we have thus far identified and quantified the expression of greater than 125 proteins from control and camptothecin-treated mouse cortical neurons. While proteins from most functional classes of proteins were identified, a particularly large percentage of the enzymes involved in glycolysis and the tricarboxylic acid cycle were observed.     

Toward a high-throughput approach to quantitative proteomic analysis: Expression-dependent protein identification by mass spectrometry

The isotope-coded affinity tag (ICAT) [1] technology enables the concurrent identification and comparative quantitative analysis of proteins present in biological samples such as cell and tissue extracts and biological fluids by mass spectrometry. The initial implementation of this technology was based on microcapillary chromatography coupled on-line with electrospray ionization tandem mass spectrometry. This implementation lacked the ability to select proteins for identification based on their relative abundance and therefore to focus on differentially expressed proteins. In order to improve the sample throughput of this technology, we have developed a two-step approach that is focused on those proteins for which the abundance changes between samples: First, a new software program for the automated quantification of ICAT reagent labeled peptides analyzed by microcapillary electrospray ionization time-of-flight mass spectrometry determines those peptides that differ in their abundance and second, these peptides are identified by tandem mass spectrometry using an electrospray quadrupole time-of flight mass spectrometer and sequence database searching. Results from the application of this approach to the analysis of differentially expressed proteins secreted from nontumorigenic human prostate epithelial cells and metastatic cancerous human prostate epithelial cells are shown.     

The impact of chromatography and mass spectrometry on the analysis of protein phosphorylation sites

Protein phosphorylation analysis is an enormous challenge. This review summarises the currently used techniques, which are based on radiolabelling and mass spectrometry as well as electrophoretic and chromatographic separation. Many methods exist, but there is still no single procedure applicable to all phosphoproteins. MS is able to deliver information about the location of phosphorylation sites, but phosphospecific properties with respect to ionisation present obstacles. Therefore, multidimensional approaches involving several analytical methods are often necessary to conquer phosphorylation site identification.Abbreviations 2D Two-dimensional - CE Capillary electrophoresis - CID Collision-induced dissociation - ECD Electron capture dissociation - ESI Electrospray ionisation - FT-ICR Fourier transform ion cyclotron resonance - HPLC High performance liquid chromatography - ICAT Isotope coded affinity tags - ICP Inductively-coupled plasma - IDA Immino-diacetic acid - IMAC Immobilised metal affinity chromatography - IRMPD Infrared multiphoton dissociation - IT Ion trap - MALDI Matrix-assisted laser desorption/ionisation - MRP14 Myeloid-related protein 14 - MS Mass spectrometry - NTA Nitrilo-triacetic acid - PAGE Polyacrylamide gel electrophoresis - PDI Protein disulfide isomerase - pS Phosphoserine residue - PSD Post-source decay - pT Phosphothreonine residue - PVDF Polyvinylidene fluoride - pY Phosphotyrosine residue - Q-TOF Quadrupole-time-of-flight - RP Reversed phase - SIM Single-ion monitoring - SDS Sodium dodecyl sulfate - SORI Sustained off-resonance irradiation - TLC Thin-layer chromatography - TOF Time-of-flight An erratum to this article can be found at     

Protein-peptide affinity determination using an H/D exchange dilution strategy: Application to antigen-antibody interactions

A new methodology using hydrogen/deuterium amide exchange (HDX) to determine the binding affinity of protein-peptide interactions is reported. The method, based on our previously established approach, protein ligand interaction by mass spectrometry, titration, and H/D exchange (PLIMSTEX) [J. Am. Chem. Soc. 2003, 125, 5252–5253], makes use of a dilution strategy (dPLIMSTEX) for HDX, using the mass of the peptide ligand as readout. We employed dPLIMSTEX to study the interaction of calcium-saturated calmodulin with the opioid peptide β-endorphin as a model system; the affinity results are in good agreement with those from traditional PLIMSTEX and with literature values obtained by using other methods. We show that the dPLIMSTEX method is feasible to quantify an antigen-antibody interaction involving a 3-nitrotyrosine modified peptide in complex with a monoclonal anti-nitrotyrosine antibody. A dissociation constant in the low nanomolar range was determined, and a binding stoichiometry of antibody/peptide of 1:2 was confirmed. In addition, we determined that the epitope in the binding interface contains a minimum of five amino acids. The dPLIMSTEX approach is a sensitive and powerful tool for the quantitative determination of peptide affinities with antibodies, complementary to conventional immuno-analytical techniques.     

Effect of chronic morphine exposure on the synaptic plasma-membrane subproteome of rats: a quantitative protein profiling study based on isotope-coded affinity tags and liquid chromatography/mass spectrometry

The effect of chronic morphine exposure on the synaptic plasma-membrane subproteome in rats was studied by the isotope-coded affinity tag (ICAT) method coupled with capillary reversed-phase liquid chromatography/electrospray ionization mass spectrometry and tandem mass spectrometry. ICAT-labeled tryptic peptides of synaptic membrane proteins were successfully identified using tandem mass spectrometry in conjunction with protein database searching. Several important synaptic plasma-membrane proteins displayed significant regulation changes as a result of chronic morphine exposure in vivo. In particular, an integral membrane protein Na(+)/K+ ATPase (alpha-subunit) involved in regulation of the cell membrane potential by controlling sodium and potassium ion permeability was downregulated by 39 +/- 2%. This result was in excellent agreement with the reduction in electrogenic Na+, K+ pumping due to about 40% downregulation of Na(+)/K+ ATPase alpha3-isoform in myenteric S-neurons of morphine-exposed guinea-pigs measured by others via immunohistochemistry. The decrease in the abundance of non-erythroid alpha II-spectrin in the synaptic plasma-membrane fraction was also observed, which was hypothetically associated with the breakdown of the protein due to the upregulation of the proteolytic enzyme caspase-3 upon chronic morphine exposure.     

Membrane-introduction mass spectrometry (MIMS)

Membrane-introduction mass spectrometry (MIMS) for chemical analysis involves directly sampling analytes in gaseous, liquid and solid samples through a semi-permeable membrane coupled to a mass spectrometer, yielding selective and sensitive quantitation. Because MIMS is an on-line technique, in which samples can be continuously flowed over a membrane interface, it can yield analytical results in real time without the need for sample clean-up and chromatographic separation. This review highlights trends and developments in MIMS over the past decade and describes recent studies that pertain to its use for on-site, in-situ and in-vivo chemical analysis. We report on advancements in instrumentation, including membrane materials, interface configurations and ionization techniques that have extended the range of analytes amenable to MIMS.We summarize the progress made in the miniaturization of mass spectrometers that have resulted in field-portable systems and review recent applications of continuous mobile monitoring and on-site environmental monitoring to yield both temporally and spatially resolved quantitative and semi-quantitative data. Finally, we describe recent work involving the use of MIMS for in-vivo chemical analysis.     

Tagless extraction-retentate chromatography: a new global protein digestion strategy for monitoring differential protein expression

A new global protein digestion and selective peptide extraction strategy for the purpose of monitoring differential protein expression, coined as tagless extraction-retentate chromatography, is introduced. Target protein populations are firstly digested under reduced and alkylated conditions, and resultant peptides selectively extracted via covalent attachment to methionine residues by bromoacetyl reactive groups tethered to the surface of glass beads packed in small reaction vessels. After conjugation, reactive beads are stringently washed to remove nonspecifically bound peptides and then later treated with beta-mercaptoethanol to release captured methionine peptides in their nascent state, without complicating affinity tags. Recovered methionine containing peptides are profiled using the surface-enhanced laser desorption/ionization (SELDI) retentate chromatography mass spectrometry (RCMS) method. Selected peptides are further studied employing ProteinChip tandem mass spectrometry (MS/MS) analysis to identify their parent proteins. This approach has been applied to an Escherichia coli lysate model system and has demonstrated facility in reducing global digest complexity, sensitivity to low protein expression levels, and significant quantitative capability. It is envisioned that tagless extraction-RCMS will evolve to be a valuable approach for both basic research and clinical proteomics endeavors.