Tissue proteomics using chemical immobilization and mass spectrometry

Proteomics analysis is important for characterizing tissues to gain biological and pathological insights, which could lead to the identification of disease-associated proteins for disease diagnostics or targeted therapy. However, tissues are commonly embedded in optimal cutting temperature medium (OCT) or are formalin-fixed and paraffin-embedded (FFPE) in order to maintain tissue morphology for histology evaluation. Although several tissue proteomic analyses have been performed on FFPE tissues using advanced mass spectrometry (MS) technologies, high-throughput proteomic analysis of OCT-embedded tissues has been difficult due to the interference of OCT in the MS analysis. In addition, molecules other than proteins present in tissues further complicate tissue proteomic analysis. Here, we report the development of a method using chemical immobilization of proteins for peptide extraction (CIPPE). In this method, proteins are chemically immobilized onto a solid support; interferences from tissues and OCT embedding are removed by extensive washing of proteins conjugated on the solid support. Peptides are then released from the solid phase by proteolysis, enabling MS analysis. This method was first validated by eliminating OCT interference from a standard protein, human serum albumin, where all of the unique peaks contributed by OCT contamination were eradicated. Finally, this method was applied for the proteomic analysis of frozen and OCT-embedded tissues using iTRAQ (isobaric tag for relative and absolute quantitation) labeling and two-dimensional liquid chromatography tandem mass spectrometry. The data showed reproducible extraction and quantitation of 10,284 proteins from 3996 protein groups and a minimal impact of OCT embedding on the analysis of the global proteome of the stored tissue samples.     

Protein profiling by the combination of two independent mass spectrometry techniques

Protein profiling in the high-throughput mode is a most useful technique that allows formation of reference databases for cells and tissues and performance of comparative proteomics. In the proposed protocol protein extraction from tissues is followed by 2D gel electrophoresis (2DE) with subsequent in-gel digestion and identification of soluble proteins by two individual mass spectrometric techniques, tandem matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and nano-liquid chromatography (nano-LC)-MS/MS. The proposed combined use of these two MS approaches leads to a very high identification rate of well-separated protein spots from a gel. In the first step 2DE separates high-abundance proteins (those visualized by nonsensitive Coomassie blue staining) that are subsequently picked, digested and aliquoted for MS applications. Protein samples not identified by MALDI-MS or MS/MS (77% of all spots) are finally unambiguously identified by nano-LC-MS/MS (total identification rate 94%). This protocol can be completed in 6 weeks.     

Relative Quantification of Proteins in Formalin-Fixed Paraffin-Embedded Breast Cancer Tissue Using Multiplexed Mass Spectrometry Assays

The identification of clinically relevant biomarkers represents an important challenge in oncology. This problem can be addressed with biomarker discovery and verification studies performed directly in tumor samples using formalin-fixed paraffin-embedded (FFPE) tissues. However, reliably measuring proteins in FFPE samples remains challenging. Here, we demonstrate the use of liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM/MS) as an effective technique for such applications. An LC-MRM/MS method was developed to simultaneously quantify hundreds of peptides extracted from FFPE samples and was applied to the targeted measurement of 200 proteins in 48 triple-negative, 19 HER2-overexpressing, and 20 luminal A breast tumors. Quantitative information was obtained for 185 proteins, including known markers of breast cancer such as HER2, hormone receptors, Ki-67, or inflammation-related proteins. LC-MRM/MS results for these proteins matched immunohistochemistry or chromogenic in situ hybridization data. In addition, comparison of our results with data from the literature showed that several proteins representing potential biomarkers were identified as differentially expressed in triple-negative breast cancer samples. These results indicate that LC-MRM/MS assays can reliably measure large sets of proteins using the analysis of surrogate peptides extracted from FFPE samples. This approach allows to simultaneously quantify the expression of target proteins from various pathways in tumor samples. LC-MRM/MS is thus a powerful tool for the relative quantification of proteins in FFPE tissues and for biomarker discovery.     

胃癌及癌旁组织定量比较蛋白质组学研究

为寻找胃癌特异的肿瘤标记物,用于胃癌临床诊断及药物治疗靶点的选择,本研究采用荧光差异显示凝胶电泳（DIGE）技术分离并筛选 Cy3、Cy5 及 Cy2 荧光素标记的胃癌及对应癌旁组织差异表达蛋白质,用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）或串联质谱技术进行鉴定并分析。结果共筛选出 33 个差异表达蛋白质点,其中 9 个蛋白质点在胃癌组织中上调,24 个蛋白质点下调。对 22 个蛋白质点采用质谱技术成功鉴定,突变结蛋白、锰超氧化物歧化酶、热休克蛋白 60等在胃癌中高表达,热休克蛋白 27、前列腺素 F 合酶、硒结合蛋白 1、锌指蛋白 160、微管蛋白 α6、真核生物翻译延伸因子 1 α1 等在胃癌组织中低表达,并筛选出 5 个未知蛋白。这些差异表达蛋白可望成为胃癌诊断的特异标记物,并与胃癌的发生、发展及预后等有关,为胃癌的诊断、发生机制的研究提供了新的思路。     

Analysis of posttranslational modifications of proteins by tandem mass spectrometry

Protein activity and turnover is tightly and dynamically regulated in living cells. Whereas the three-dimensional protein structure is predominantly determined by the amino acid sequence, posttranslational modification (PTM) of proteins modulates their molecular function and the spatial-temporal distribution in cells and tissues. Most PTMs can be detected by protein and peptide analysis by mass spectrometry (MS), either as a mass increment or a mass deficit relative to the nascent unmodified protein. Tandem mass spectrometry (MS/MS) provides a series of analytical features that are highly useful for the characterization of modified proteins via amino acid sequencing and specific detection of posttranslationally modified amino acid residues. Large-scale, quantitative analysis of proteins by MS/MS is beginning to reveal novel patterns and functions of PTMs in cellular signaling networks and biomolecular structures.     

Identification of Protein Markers Specific for Papillary Renal Cell Carcinoma Using Imaging Mass Spectrometry

Since the emergence of proteomics methods, many proteins specific for renal cell carcinoma (RCC) have been identified. Despite their usefulness for the specific diagnosis of RCC, such proteins do not provide spatial information on the diseased tissue. Therefore, the identification of cancer-specific proteins that include information on their specific location is needed. Recently, matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) based imaging mass spectrometry (IMS) has emerged as a new tool for the analysis of spatial distribution as well as identification of either proteins or small molecules in tissues. In this report, surgical tissue sections of papillary RCC were analyzed using MALDI-IMS. Statistical analysis revealed several discriminative cancer-specific m/z-species between normal and diseased tissues. Among these m/z-species, two particular proteins, S100A11 and ferritin light chain, which are specific for papillary RCC cancer regions, were successfully identified using LC-MS/MS following protein extraction from independent RCC samples. The expressions of S100A11 and ferritin light chain were further validated by immunohistochemistry of human tissues and tissue microarrays (TMAs) of RCC. In conclusion, MALDI-IMS followed by LC-MS/MS analysis in human tissue identified that S100A11 and ferritin light chain are differentially expressed proteins in papillary RCC cancer regions.     

Quantitative profile of five murine core proteomes using label-free functional proteomics

Analysis of primary animal and human tissues is key in biological and biomedical research. Comparative proteomics analysis of primary biological material would benefit from uncomplicated experimental work flows capable of evaluating an unlimited number of samples. In this report we describe the application of label-free proteomics to the quantitative analysis of five mouse core proteomes. We developed a computer program and normalization procedures that allow exploitation of the quantitative data inherent in LC-MS/MS experiments for relative and absolute quantification of proteins in complex mixtures. Important features of this approach include (i) its ability to compare an unlimited number of samples, (ii) its applicability to primary tissues and cultured cells, (iii) its straightforward work flow without chemical reaction steps, and (iv) its usefulness not only for relative quantification but also for estimation of absolute protein abundance. We applied this approach to quantitatively characterize the most abundant proteins in murine brain, heart, kidney, liver, and lung. We matched 8,800 MS/MS peptide spectra to 1,500 proteins and generated 44,000 independent data points to profile the approximately 1,000 most abundant proteins in mouse tissues. This dataset provides a quantitative profile of the fundamental proteome of a mouse, identifies the major similarities and differences between organ-specific proteomes, and serves as a paradigm of how label-free quantitative MS can be used to characterize the phenotype of mammalian primary tissues at the molecular level.     

Protein expression profiling of endometrial malignancies reveals a new tumor marker: chaperonin 10

Endometrial carcinoma is a common malignancy in women, being exceeded in incidence only by that of breast, lung, and colorectal cancers. At present, no serum tumor markers are available for the monitoring of endometrial carcinoma patients, and patients with recurrent disease are detected only following the development of symptoms or abnormalities in imaging assessments. Similarly, no screening tools are available for endometrial carcinoma. Protein profiling by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has proven to be a sensitive and fast method of analysis for small proteins or peptides to yield specific biomarkers. In this study, a variety of normal and malignant endometrial tissue samples were fractionated and analyzed by SELDI-TOF MS (SELDI is a version of MALDI utilizing protein "chips"). A number of proteins displayed differential expression in malignant endometrial tissues. One of the prominent proteins fractionated by weak cation exchange chromatography and displaying enhanced expression in these malignant tissues was identified as chaperonin 10. The increased expression of chaperonin 10 in malignant endometrial tissues was further confirmed by parallel Western blot and immunohistochemistry analyses.     

肺鳞癌患者与健康人血清的差异蛋白质组学研究

为筛选肺鳞癌的血清标志物,采用二维凝胶电泳(2-DE)技术分离I期肺鳞癌患者和健康人的血清蛋白质,PDquest图像分析软件识别差异蛋白质点,电喷雾串联质谱(ESI-Q-TOF MS/MS)鉴定差异蛋白,然后应用蛋白质印迹和免疫组化方法分别检测差异蛋白——结合珠蛋白-2(haptoglobin-2,HP-2)在肺鳞癌患者血清和健康人血清以及肺鳞癌组织和癌旁正常支气管上皮组织中的表达.建立了肺鳞癌患者和健康人血清的2-DE图谱,图像分析软件识别了1O个差异蛋白质点,质谱鉴定了4种差异蛋白;蛋白质印迹分析显示,HP-2在肺鳞癌血清中的表达水平显著高于健康人(P<0.05),但其表达水平与肺鳞癌的临床分期无明显相关性;免疫组化结果显示,HP-2在肺鳞癌组织中的表达水平高于癌旁正常支气管上皮组织(P<0.05).研究结果提示:HP-2是候选的肺鳞癌血清分子标志物,血清中HP-2水平对肺鳞癌诊断可能具有一定的参考价值;肺鳞癌组织中HP-2表达上调可能是患者血清中HP-2表达升高的原因之一.     

Protein recovery and identification from the gulf killifish, Fundulus grandis: comparing snap-frozen and RNAlater® preserved tissues

Reliable proteomic analysis of biological tissues requires sampling approaches that preserve proteins as close to their in vivo state as possible. In the current study, the patterns of protein abundance in one‐dimensional (1‐D) gels were assessed for five tissues of the gulf killifish, Fundulus grandis, following snap‐freezing tissues in liquid nitrogen or immersion of fresh tissues in RNAlater^®. In liver and heart, the protein profiles in 1‐D gels were better preserved by snap‐freezing, while in gill, the 1‐D protein profile was better preserved by immersion in RNAlater^®. In skeletal muscle and brain, the two approaches yielded similar patterns of protein abundance. LC‐MS/MS analyses and database searching resulted in the identification of 17 proteins in liver and 12 proteins in gill. Identified proteins include enzymes of energy metabolism, structural proteins, and proteins serving other biological functions. These protein identifications for a species without a sequenced genome demonstrate the utility of F. grandis as a model organism for environmental proteomic studies in vertebrates.     

Decreased activity of the 20S proteasome in the brain white matter and gray matter of patients with multiple sclerosis

Carbonylated (oxidized) proteins are known to accumulate in the cerebral white matter (WM) and gray matter (GM) of patients with multiple sclerosis (MS). Although oxidative stress is necessary for carbonyl generation, it is the failure of the degradation systems that ultimately leads to the build-up of carbonylated proteins within tissues. In this study, we measured the activity of the 20S proteasome and other proteolytic systems in the cerebral WM and GM of 13 MS patients and 13 controls. We report that the activities of the three peptidases of the 20S proteasome (i.e. chymotrypsin-like, caspase-like and trypsin-like) in both MS-WM and MS-GM are greatly reduced. Interestingly, neither the amount of proteasome nor the levels of the catalytic subunits (β1, β2, and β5) are diminished in this disease. Proteins containing Lys-48 poly-ubiquitin also accumulate in MS tissues, indicating failure of the 26S proteasome as well. Levels of the regulatory caps 11S α and 19S are also lower in MS than in controls, suggesting that the activity of the more complex proteasomes may be reduced further. Finally, the activities of other proteases that might also remove oxidized proteins (calpain, cathepsin B, mitochondrial LonP) are not lessened in MS. Together, these studies suggest that direct inactivation of proteolytic centers in the 20S particle and/or the presence of specific inhibitors is the underlying cause of proteasomal dysfunction in MS.     

Proteome analysis of embryo and endosperm from germinating tomato seeds

Proteome analysis of embryo and endosperm tissues from germinating tomato seed was conducted using 1-DE, 2-DE, and MS. Mobilization of the most abundant proteins, which showed similar profiles in the two tissues, occurred first in the endosperm. CBB R-250 staining of 2-DE gels revealed 352 and 369 major protein spots in the embryo and endosperm, respectively, at 0 h. Of these, 75 major spots were selected, excised, in-gel digested with trypsin, and analyzed by MALDI-TOF-MS and/or LC-ESI-Q/TOF-MS/MS. Peptide MS and MS/MS data were searched against publicly available protein and EST databases, and 47 proteins identified. Embryo-specific proteins included a BAC19.13 homologue, whereas four proteins specific to the endosperm were tomato mosaic virus coat proteins related to defense mechanisms. The most abundant proteins both in the embryo and endosperm were seed storage proteins, i.e., legumins (11 spots), vicilins (11 spots), albumin (2 spots). Housekeeping enzymes, actin-binding profilin, defense-related protein kinases, nonspecific lipid transfer protein, and proteins involved in general metabolism were also identified. The roles of some of the proteins identified in the embryo and endosperm are discussed in relation to seed germination in tomato.     

Proteome profiling of human epithelial ovarian cancer cell line TOV-112D

A proteome profiling of the epithelial ovarian cancer cell line TOV-112D was initiated as a protein expression reference in the study of ovarian cancer. Two complementary proteomic approaches were used in order to maximise protein identification: two-dimensional gel electrophoresis (2DE) protein separation coupled to matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and one-dimensional gel electrophoresis (1DE) coupled to liquid-chromatography tandem mass spectrometry (LC MS/MS). One hundred and seventy-two proteins have been identified among 288 spots selected on two-dimensional gels and a total of 579 proteins were identified with the 1DE LC MS/MS approach. This proteome profiling covers a wide range of protein expression and identifies several proteins known for their oncogenic properties. Bioinformatics tools were used to mine databases in order to determine whether the identified proteins have previously been implicated in pathways associated with carcinogenesis or cell proliferation. Indeed, several of the proteins have been reported to be specific ovarian cancer markers while others are common to many tumorigenic tissues or proliferating cells. The diversity of proteins found and their association with known oncogenic pathways validate this proteomic approach. The proteome 2D map of the TOV-112D cell line will provide a valuable resource in studies on differential protein expression of human ovarian carcinomas while the 1DE LC MS/MS approach gives a picture of the actual protein profile of the TOV-112D cell line. This work represents one of the most complete ovarian protein expression analysis reports to date and the first comparative study of gene expression profiling and proteomic patterns in ovarian cancer.     

Genome-scale identification of membrane-associated human mRNAs

The subcellular localization of proteins is critical to their biological roles. Moreover, whether a protein is membrane-bound, secreted, or intracellular affects the usefulness of, and the strategies for, using a protein as a diagnostic marker or a target for therapy. We employed a rapid and efficient experimental approach to classify thousands of human gene products as either "membrane-associated/secreted" (MS) or "cytosolic/nuclear" (CN). Using subcellular fractionation methods, we separated mRNAs associated with membranes from those associated with the soluble cytosolic fraction and analyzed these two pools by comparative hybridization to DNA microarrays. Analysis of 11 different human cell lines, representing lymphoid, myeloid, breast, ovarian, hepatic, colon, and prostate tissues, identified more than 5,000 previously uncharacterized MS and more than 6,400 putative CN genes at high confidence levels. The experimentally determined localizations correlated well with in silico predictions of signal peptides and transmembrane domains, but also significantly increased the number of human genes that could be cataloged as encoding either MS or CN proteins. Using gene expression data from a variety of primary human malignancies and normal tissues, we rationally identified hundreds of MS gene products that are significantly overexpressed in tumors compared to normal tissues and thus represent candidates for serum diagnostic tests or monoclonal antibody-based therapies. Finally, we used the catalog of CN gene products to generate sets of candidate markers of organ-specific tissue injury. The large-scale annotation of subcellular localization reported here will serve as a reference database and will aid in the rational design of diagnostic tests and molecular therapies for diverse diseases.     

Discovery of regulatory molecular events and biomarkers using 2D capillary chromatography and mass spectrometry

An important component of proteomic research is the high-throughput discovery of novel proteins and protein-protein interactions that control molecular events that contribute to critical cellular functions and human disease. The interactions of proteins are essential for cellular functions. Identifying perturbation of normal cellular protein interactions is vital for understanding the disease process and intervening to control the disease. A second area of proteomics research is the discovery of proteins that will serve as biomarkers for the early detection, diagnosis and drug treatment response for specific diseases. These studies have been referred to as clinical proteomics. To discover biomarkers, proteomics research employs the quantitative comparison of peptide and protein expression in body fluids and tissues from diseased individuals (case) versus normal individuals (control). Methods that couple 2D capillary liquid chromatography (LC) and tandem mass spectrometry (MS/MS) analysis have greatly facilitated this discovery science. Coupling 2D-LC/MS/MS analysis with automated genome-assisted spectra interpretation allows a direct, high-throughput and high-sensitivity identification of thousands of individual proteins from complex biological samples. The systematic comparison of experimental conditions and controls allows protein function or disease states to be modeled. This review discusses the different purification and quantification strategies that have been developed and used in combination with 2D-LC/MS/MS and computational analysis to examine regulatory protein networks and clinical samples.     

Discovery of regulatory molecular events and biomarkers using 2D capillary chromatography and mass spectrometry

An important component of proteomic research is the high-throughput discovery of novel proteins and protein–protein interactions that control molecular events that contribute to critical cellular functions and human disease. The interactions of proteins are essential for cellular functions. Identifying perturbation of normal cellular protein interactions is vital for understanding the disease process and intervening to control the disease. A second area of proteomics research is the discovery of proteins that will serve as biomarkers for the early detection, diagnosis and drug treatment response for specific diseases. These studies have been referred to as clinical proteomics. To discover biomarkers, proteomics research employs the quantitative comparison of peptide and protein expression in body fluids and tissues from diseased individuals (case) versus normal individuals (control). Methods that couple 2D capillary liquid chromatography (LC) and tandem mass spectrometry (MS/MS) analysis have greatly facilitated this discovery science. Coupling 2D-LC/MS/MS analysis with automated genome-assisted spectra interpretation allows a direct, high-throughput and high-sensitivity identification of thousands of individual proteins from complex biological samples. The systematic comparison of experimental conditions and controls allows protein function or disease states to be modeled. This review discusses the different purification and quantification strategies that have been developed and used in combination with 2D-LC/MS/MS and computational analysis to examine regulatory protein networks and clinical samples.     

Proteomic analysis of proteins from bronchoalveolar lavage fluid reveals the action mechanism of ultrafine carbon black-induced lung injury in mice

Previous studies have shown that ultrafine carbon black (ufCB) could cause oxidative stress and lung injury, but the mechanisms have not been clearly demonstrated. In this study, 1-D gel electrophoresis coupled with LC/MS/MS (1-D geLC/MS/MS) was carried out with bronchoalveolar lavage fluid (BALF) to identify proteins associated with ufCB-induced lung injury. If required, Western blot was conducted additionally to validate proteins. Thirty-three proteins were identified, including leukemia inhibitory factor receptor (LIFR) and epidermal growth factor receptor (EGFR). Western blot analysis showed that ufCB exposure caused the increases of LIFR and EGFR in BALF and decreases of both receptors in lung tissues, suggesting the acceleration of epithelial shedding from the lung and increase of cell debris with membrane proteins EGFR and LIFR in BALF. There were strong correlations between vascular endothelial growth factor (VEGF) and albumin (p<0.01) or alpha2-macroglobulin (alpha2M) in BALF (p<0.05). Importantly, antioxidant ceruloplasmin (Cp) was shown to be produced from lung epithelial cells in response to ufCB exposure. This is the first study to apply 1-D ge LC/MS/MS and experimental studies to reveal the mechanisms involved in the pathogenesis of ufCB-induced lung injury.     

2‐D PAGE and MS analysis of proteins from formalin‐fixed,paraffin‐embedded tissues

In the past decade, encouraging results have been obtained in extraction and analysis of proteins from formalin‐fixed, paraffin‐embedded (FFPE) tissues. However, 2‐D PAGE protein maps with satisfactory proteomic information and comparability to fresh tissues have never been described to date. In the present study, we report 2‐D PAGE separation and MS identification of full‐length proteins extracted from FFPE skeletal muscle tissue. The 2‐D protein profiles obtained from FFPE tissues could be matched to those achieved from frozen tissues replicates. Up to 250 spots were clearly detected in 2‐D maps of proteins from FFPE tissue following standard mass‐compatible silver staining. Protein spots from both FFPE and frozen tissue 2‐D gels were excised, subjected to in situ hydrolysis, and identified by MS analysis. Matched spots produced matched protein identifications. Moreover, 2‐D protein maps from FFPE tissues were successfully subjected to Western immunoblotting, producing comparable results to fresh‐frozen tissues. In conclusion, this study provides evidence that, when adequately extracted, full‐length proteins from FFPE tissues might be suitable to 2‐D PAGE‐MS analysis, allowing differential proteomic studies on the vast existing archives of healthy and pathological‐fixed tissues.     

Functional consequences of actin nitration: in vitro and in disease states

To link the phenomena of inflammatory-induced increases in protein nitrotyrosine (NO₂Tyr) derivatives to protein dysfunction and consequent pathological conditions, the evaluation of discrete NO₂Tyr modifications on specific proteins must be undertaken. Mass spectrometric (MS) proteomics-based strategies allow for the identification of all individual proteins that are nitrated by separating tissue homogenates using 2D gel electrophoresis, detecting the nitrated proteins using an anti-NO₂Tyr antibody, and then identifying the peptides generated during an in-gel proteolytic digest using matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) MS. Actin, one of the most abundant proteins in eukaryotic cells, constitutes 5% or more of cell protein and serves with other cytoskeletal proteins as a critical target for nitration-induced functional impairment. Herein, examples of actin nitration detected under physiological conditions in various models of human disease or in clinically derived tissues are given and the impact that this post-translational protein modification can have on cell and organ function is discussed.