Targeted protein quantitation in human body fluids by mass spectrometry.

Human body fluids (biofluids) contain various proteins, some of which reflect individuals' physiological conditions or predict diseases. Therefore, the analysis of biofluids can provide substantial information on novel biomarkers for clinical diagnosis and prognosis. In the past decades, mass spectrometry (MS)-based technologies have been developed as proteomic strategies not only for the identification of protein biomarkers but also for biomarker verification/validation in body fluids for clinical applications. The main advantage of targeted MS-based methodologies is the accurate and specific simultaneous quantitation of multiple biomarkers with high sensitivity. Here, we review MS-based methodologies that are currently used for the targeted quantitation of protein components in human body fluids, especially in plasma, urine, cerebrospinal fluid, and saliva. In addition, the currently used MS-based methodologies are summarized with a specific focus on applicable clinical sample types, MS configurations, and acquisition modes.

Quantitative Proteomics of Th-MYCN Transgenic Mice Reveals Aurora Kinase Inhibitor Altered Metabolic Pathways and Enhanced ACADM To Suppress Neuroblastoma Progression.

Neuroblastoma is a neural crest-derived embryonal tumor and accounts for about 15% of all cancer deaths in children. MYCN amplification is associated with aggressive and advanced stage of high-risk neuroblastoma, which remains difficult to treat and exhibits poor survival under current multimodality treatment. Here, we analyzed the transcriptomic profiles of neuroblastoma patients and showed that aurora kinases lead to poor survival and had positive correlation with MYCN amplification and high-risk disease. Further, pan-aurora kinase inhibitor (tozasertib) treatment not only induces cell-cycle arrest and suppresses cell proliferation, migration, and invasion ability in MYCN-amplified (MNA) neuroblastoma cell lines, but also inhibits tumor growth and prolongs animal survival in Th-MYCN transgenic mice. Moreover, we performed quantitative proteomics and identified 150 differentially expressed proteins after tozasertib treatment in the Th-MYCN mouse model. The functional and network-based enrichment revealed that tozasertib alters metabolic processes and identified a mitochondrial flavoenzyme in fatty acid ß-oxidation, ACADM, which is correlated with aurora kinases and neuroblastoma patient survival. Our findings indicate that the aurora kinase inhibitor could cause metabolic imbalance, possibly by disturbing carbohydrate and fatty acid metabolic pathways, and ACADM may be a potential target in MNA neuroblastoma.

Comparison of different fractionation strategies for in-depth phosphoproteomics by liquid chromatography tandem mass spectrometry.

Phosphorylation, a major posttranslational modification of proteins, plays an important role in protein activity and cell signaling. However, it is difficult to detect protein phosphorylation because of its low abundance and the fact that the analysis can be hindered by the presence of highly abundant non-phosphoproteins. In order to reduce the sample complexity and improve the efficiency of identification of phosphopeptides, aliphatic hydroxy acid-modified metal oxide chromatography (HAMMOC) was utilized to enrich phosphopeptides from a murine macrophage cell lysate. Strong cation chromatography (SCX), electrostatic repulsion hydrophilic interaction chromatography (ERLIC), and solution isoelectric focusing (sIEF) were investigated in detail for phosphopeptide fractionation strategies followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. A total of 5744 non-redundant phosphopeptides and 2159 phosphoproteins were identified from the cell lysates in three fractionation approaches. The SCX fractionation contained the largest number of phosphoproteins and phosphopeptides that were identified. In addition, 4336, 2064, and 2424 phosphopeptides were identified from SCX-LC-MS/MS, ERLIC-LC-MS/MS, and sIEF-LC/MS-MS, including 2430, 438, and 751 phosphopeptides that were only specifically found in SCX, ERLIC, and sIEF fractionations. In conclusion, these three fractionation strategies demonstrated great complementarity, which greatly improved the efficiency of identification of phosphopeptides and can be suitable for use in in-depth phosphoproteome research. Graphical Abstract.

Evaluation of disulfide scrambling during the enzymatic digestion of bevacizumab at various pH values using mass spectrometry.

Disulfide linkages play an important role in protein stability and activity. Thus, it is critical to characterize disulfide bonds to ensure the quality and function of protein pharmaceuticals. There are, however, problems associated with maintaining disulfide linkages in the conventional procedures that are used to digest a protein. In order to preserve enzyme activity during the digestion of a protein, it is commonly carried out at neutral to basic environment which increases the possibilities of disulfide bond scrambling. However, it is not easy to differentiate whether the scrambled disulfide linkages are initiated by the sample itself or whether they are induced during the protease digestion process. In this study, the optimum pH for minimizing disulfide bond rearrangements during the digestion process was determined. Three sets of proteases, trypsin plus Glu-C, Lys-C and thermolysin were used, followed by dimethyl labeling and mass spectrometry for a bevacizumab (Avastin) disulfide linkage analysis. No disulfide linkage scrambling was detected at pH6 when Lys-C or trypsin plus Glu-C were used as enzymes. When thermolysin was applied, some scrambled disulfide bonds were identified at pH5, 6 and 7. Nevertheless, there was less disulfide bond scrambling at a lower pH. All correct disulfide bonds on bevacizumab could be identified using this approach. The results demonstrated that by choosing the proper enzymes, using a lower pH environment for the digestion could reduce the degree of artifact disulfide scrambling.

Differential proteomics of monosodium urate crystals-induced inflammatory response in dissected murine air pouch membranes by iTRAQ technology.

The precipitation of monosodium urate crystals within joints triggers an acute inflammatory reaction that is the root cause of gout. The inflammation induced by the injection of MSU crystals into the murine air pouch for 1, 3, and 5 h was examined by iTRAQ-based proteomic profiling. The iTRAQ-labeled peptides were fractionated by SCX, basic-RP or solution-IEF, followed by LC-MS/MS analysis. A total of 951 proteins were quantified from the total combined fractions. Among them, 317 proteins exhibited a differential expression, compared to that of the controls at one time point or more. The majority of the differentially expressed proteins were found in the sample after a 5-h MSU treatment. Western blot revealed that the expression levels of cathelin-related antimicrobial peptide and S100A9 were positively correlated with the time-course treated with MSU. Further analysis of GeneGO pathway demonstrated that these differentially expressed proteins are primarily related to the immune-related complement system and the tricarboxylic acid cycle. Moreover, seven genes from the TCA cycle were found to be significantly downregulated at the transcriptional level and its correlation with gout and possible therapeutic applications are worth further investigation. Last, we found that pyruvate carboxylation could be potential targets for antigout treatment.

Quantitative analysis of prostate specific antigen isoforms using immunoprecipitation and stable isotope labeling mass spectrometry.

Prostate specific antigen (PSA) is a widely used serum marker for prostate cancer (PCa), but has limited specificity for distinguishing early PCa from benign prostatic hyperplasia (BPH). Recently, proPSAs, comprised of native proPSA, as well as truncated proPSA forms, [-2] proPSA, [-5] proPSA, and [-7] proPSA, have been shown to be better diagnostic targets than PSA for PCa. Stable isotope labeling-multiple reaction monitoring mass spectrometry (SIL/MRM-MS) has been frequently used to measure low-abundance biomarkers in tissues and biofluids, owing to its high sensitivity and specificity, simplicity, and multiplexing capability. In this study, we have developed and optimized a strategy using immunoprecipitation in conjunction with SIL/MRM-MS assay which is capable of sensitive and accurate quantification of proPSA in serum. Since serum and plasma are by far the most complex biological fluids, the immunoprecipitation workflow was optimized to achieve sufficient sensitivity, efficiencies of protein purification with immunoaffinity depletion were determined. The developed strategy can detect proPSA and PSA with a limit of detection (LOD) and limit of quantitation (LOQ) at nanogram per milliliter levels, corresponding to a concentration 6 orders-of-magnitude lower than the most abundant serum proteins. Furthermore, the simultaneous measurement of multiple biomarkers, including the mature and precursor forms of PSA, can be achieved in a single multiplexed analysis using LC/MRM-MS. The strategy demonstrated here provides an attractive alternative to ELISAs or RIAs for the reliably measurement of proPSA to improve the specificity of PCa diagnosis.

Global disulfide bond profiling for crude snake venom using dimethyl labeling coupled with mass spectrometry and RADAR algorithm.

Snake venom consists of toxin proteins with multiple disulfide linkages to generate unique structures and biological functions. Determination of these cysteine connections usually requires the purification of each protein followed by structural analysis. In this study, dimethyl labeling coupled with LC-MS/MS and RADAR algorithm was developed to identify the disulfide bonds in crude snake venom. Without any protein separation, the disulfide linkages of several cytotoxins and PLA2 could be solved, including more than 20 disulfide bonds. The results show that this method is capable of analyzing protein mixture. In addition, the approach was also used to compare native cytotoxin 3 (CTX III) and its scrambled isomer, another category of protein mixture, for unknown disulfide bonds. Two disulfide-linked peptides were observed in the native CTX III, and 10 in its scrambled form, X-CTX III. This is the first study that reports a platform for the global cysteine connection analysis on a protein mixture. The proposed method is simple and automatic, offering an efficient tool for structural and functional studies of venom proteins.

Quantification of Hop-Derived Bitter Compounds in Beer Using Liquid Chromatography Mass Spectrometry.

Hops (Humulus lupulus L.) are essential raw materials for beer brewing, and the major contributors to beer bitterness are isohumulones (iso-α-acids) and humulinones. In recent years, many breweries have focused on the production of hop-forward beer styles by adding hops after or during the cold fermentation stage, which will tend to release humulinones or other hop-derived bitter compounds. In this study, a LC-MS/MS method was developed for quantification of 60 hop-derived bitter compounds in 25 min. Reverse-phase chromatography with an alkaline methanol/acetonitrile (70:30) mobile phase was used for the separation. The quantitative range was 0.053-3912 ng/mL with correlation coefficient r > 0.99, and the LOQ were 0.26 and 0.053 ng/mL for iso-α-acids and humulinones. Precision (RSD < 5.0%) and accuracy (recovery 86.3%-118.1%) were both satisfactory. The abundance of hop-derived bitter compounds in the dry-hopped beer (Double-India Pale Ale) and the nondry-hopped beer (Vienna Lager) were monitored throughout the fermentation and storage stages, and the formation of oxidation and cyclization products showed difference profiles between these two beers. The quantification results reveal how hop-derived bitter compounds change throughout the brewing process, as well as the influence of hops and brewing techniques on beer bitterness.

Differential proteomic analysis of cancer stem cell properties in hepatocellular carcinomas by isobaric tag labeling and mass spectrometry.

Malignant tumors are relatively resistant to treatment due to their heterogeneous nature, drug resistance, and tendency for metastasis. Recent studies suggest that a subpopulation of cancer cells is responsible for the malignant outcomes. These cells are considered as cancer stem cells (CSC). Although a number of molecules have been identified in different cancer cells as markers for cancer stem cells, no promising markers are currently available for hepatocellular carcinoma cells. In this study, two clones of Hep3B cell lines were functionally characterized as control or CSC-like cells, based on properties including spheroid formation, drug resistance, and tumor initiation. Furthermore, their protein expression profiles were investigated by isobaric tags for relative and absolute quantitation (iTRAQ), and a total of 1,127 proteins were identified and quantified from the combined fractions; 50 proteins exhibited at least 2-fold differences between these two clones. These 50 proteins were analyzed by GeneGo and were found to be associated with liver neoplasms, hepatocellular carcinoma (HCC), and liver diseases. They were also components of metabolic pathways, immune responses, and cytoskeleton remodeling. Among these proteins, the expressions of S100P, S100A14, and vimentin were verified in several HCC cell lines, and their expressions were correlated with tumorigenicity in HCC cell lines. The functional significance of vimentin and S100A14 were also investigated and verified.

Automatic disulfide bond assignment using a1 ion screening by mass spectrometry for structural characterization of protein pharmaceuticals.

An automatic method for disulfide bond assignment using dimethyl labeling and computational screening of a(1) ions with customized software, RADAR, is developed. By utilization of the enhanced a(1) ions generated from labeled peptides, the N-terminal amino acids from disulfide-linked peptides can be determined. In this study, we applied this method for structural characterization of recombinant monoclonal antibodies, an important group of therapeutic proteins. In addition to a(1) ion screening and molecular weight match, new RADAR is capable of confirming the matched peptide pairs by further comparing the collision-induced dissociation (CID) fragment ions. With the N-terminal amino acid identities as a threshold, the identification of disulfide-linked peptide pairs can be achieved rapidly at a higher confidence level. Unlike most current approaches, prior knowledge of disulfide linkages or a high-end mass spectrometer is not required, and tedious work or deliberate interpretation can be avoided in this study. Our approach makes it possible to analyze unknown disulfide bonds of protein pharmaceuticals as well as their degraded forms without further protein separation. It can be used as a convenient quality examination tool during biopharmaceutical development and manufacturing processes.

Evaluation of peptide fractionation strategies used in proteome analysis.

Peptide fractionation is extremely important for the comprehensive analysis of complex protein mixtures. Although a few comparisons of the relative separation efficiencies of 2-D methodologies using complex biological samples have appeared, a systematic evaluation was conducted in this study. Four different fractionation methods, namely strong-cation exchange, hydrophilic interaction chromatography, alkaline-RP and solution isoelectric focusing, which can be used prior to LC-MS/MS analysis, were compared. Strong-cation exchange × RPLC was used after desalting the sample; significantly more proteins were identified, compared with the nondesalted sample (1990 and 1375). We also found that the use of a combination of analytical methods resulted in a dramatic increase in the number of unique peptides that could be identified, compared with only a small increase in protein levels. The increased number of distinct peptides that can be identified is especially beneficial, not only for unequivocally identifying proteins but also for proteomic studies involving posttranslational modifications and peptide-based quantification approaches using stable isotope labeling. The identification and quantification of more peptides per protein provide valuable information that improves both the quantification of, and confidence of protein identification.

Analysis of highly polar pesticides in foods by LC-MS/MS.

Highly polar pesticides (HPP) are a group of pesticides that are characterize as low Log Kow. Many high-throughput multi-residue analysis methods based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of such polar pesticides have been proposed. In this article, we summarize the various sample preparation methods including quick polar pesticides (QuPPe), dispersive solid phase extraction (dSPE), solid phase extraction (SPE) and QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe), especially for QuPPe, which are mainly used for the determination of HPP in foods. In addition, we summarize LC-based separation methodologies that are currently used for the analysis of HPP in foods, including reversed-phase chromatography (RPC), hydrophilic interaction liquid chromatography (HILIC), ion chromatography (IC) and mixed-mode chromatography (MMC). Finally, the current mass spectrometry-based methodologies for the analysis of HPP are summarized with a specific focus on MS configurations and acquisition modes.

The use of atmospheric-pressure chemical ionization for pesticide analysis using liquid chromatography mass spectrometry.

Based on the regulations of the Ministry of Health and Welfare (MOHW) of Taiwan in 2017, an analysis of 373 pesticides in food was conducted using the MOHW official method. The analyses involved the use of either liquid chromatography mass spectrometry (LC-MS) with electrospray ionization (ESI) or gas chromatography mass spectrometry (GC-MS) with electron ionization (EI). In this study, the applicability of detecting pesticides using atmospheric pressure chemical ionization (APCI) was investigated and evaluated. The pesticides were separated using an aqueous solution of ammonium formate with methanol as the mobile phase, and ionization efficiency was compared between APCI, ESI, and EI coupled with triple quadrupole mass spectrometer using multiple reaction monitoring (MRM) acquisition. Among the 196 pesticides that were originally analyzed by ESI, 164 could be successfully detected by APCI with 6 showing a higher sensitivity when APCI was used. Among the 177 pesticides that were analyzed by EI, 43 could be successfully detected by APCI. The results also showed that APCI gave superior ionization efficiency for pesticides containing triazine, imidazole, triazole, and pyrazole groups.

Oncogenic Roles of Polycomb Repressive Complex 2 in Bladder Cancer and Upper Tract Urothelial Carcinoma.

Cancers of the urinary tract are one of the most common malignancies worldwide, causing high morbidity and mortality, and representing a social burden. Upper tract urothelial carcinoma (UTUC) accounts for 5−10% of urinary tract cancers, and its oncogenic mechanisms remain elusive. We postulated that cancers of the lower and the upper urinary tract may share some important oncogenic mechanisms. Therefore, the oncogenic mechanisms discovered in the lower urinary tract may guide the investigation of molecular mechanisms in the upper urinary tract. Based on this strategy, we revisited a high-quality transcriptome dataset of 510 patients with non-muscle invasive bladder cancer (NMIBC), and performed an innovative gene set enrichment analysis of the transcriptome. We discovered that the epigenetic regulation of polycomb repressive complex 2 (PRC2) is responsible for the recurrence and progression of lower-track urinary cancers. Additionally, a PRC2-related gene signature model was discovered to be effective in classifying bladder cancer patients with distinct susceptibility of subsequent recurrence and progression (log-rank p < 0.001 and = 0.001, respectively). We continued to discover that the same model can differentiate stage T3 UTUC patients from stage Ta/T1 patients (p = 0.026). Immunohistochemical staining revealed the presence of PRC2 components (EZH2, EED, and SUZ12) and methylated PRC2 substrates (H3K27me3) in the archived UTUC tissues. The H3K27me3 exhibited higher intensity and area intensity product in stage T3 UTUC tissues than in stage Ta/T1 tissues (p = 0.006 and 0.015, respectively), implicating stronger PRC2 activity in advanced UTUC. The relationship between H3K27 methylation and gene expression is examined using correlations. The H3K27me3 abundance is positively correlated with the expression levels of CDC26, RP11-2B6, MAPK1IP1L, SFR1, RP11-196B3, CDK5RAP2, ANXA5, STX11, PSMD5, and FGFRL1. It is also negatively correlated with CNPY2, KB-1208A12, RP11-175B9, ZNF692, RANP8, RP11-245C17, TMEM266, FBXW9, SUGT1P2, and PRH1. In conclusion, PRC2 and its epigenetic effects are major oncogenic mechanisms underlying both bladder cancer and UTUC. The epigenetically regulated genes of PRC2 in urothelial carcinoma were also elucidated using correlation statistics.

Analysis of heterocyclic aromatic amines using selective extraction by magnetic molecularly imprinted polymers coupled with liquid chromatography - Mass spectrometry.

Heterocyclic aromatic amines (HCAs) are highly carcinogenic and mutagenic chemicals. This study reports on the development of magnetic molecularly imprinted polymers (MMIPs) for the purification and quantification of HCAs. A novel magnetic molecularly imprinted polymer was successfully prepared using a surface molecular imprinting method using functionalized Fe particles as the magnetic cores. 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) was used as a molecular template; methacrylic acid (MAA), ethylene glycol dimethyl acrylate (EGDMA), 2, 2'-Azobis (2-methylpropionitrile) were used as the functional monomer, crosslinker, and initiator, respectively. The use of the template/functional monomer/crosslinking agent at a ratio of 1:4:20 resulted in a product with better adsorption properties (3.24 mg/g). The HCAs were successfully detected and quantified in processed meat samples by MISPE and LC-MS/MS. Under the final optimized detection conditions, the proposed method offered good linearity (R > 0.995) for the five HCAs with an acceptable level of precision, and an LOQ of 0.05 ng/g was successfully achieved.

Tenofovir Hampers the Efficacy of Sorafenib in Prolonging Overall Survival in Hepatocellular Carcinoma.

Sorafenib is a first-line treatment for patients with advanced hepatocellular carcinoma (HCC). These patients may simultaneously receive anti-hepatitis B treatment if they are viremic. The N-Acetylgalactosaminyltransferase 14 (GALNT14) gene can serve as a biomarker to guide HCC treatments. However, the enzyme substrates of its gene product, GalNAc-T14 (a glycosyltransferase), remained uncharacterized. Here, we conducted a glycoproteome-wide search for GalNAc-T14 substrates using lectin affinity chromatography followed by tandem mass spectrometry. Seventeen novel GalNAc-T14 substrates were identified. A connective map analysis showed that an antiviral drug, tenofovir, was the leading medicinal compound to down-regulate the expression of these substrates. In vitro assays showed that HCC cells were resistant to sorafenib if pretreated by tenofovir but not entecavir. Clinical analysis showed that the concomitant use of tenofovir and sorafenib was a previously unrecognized predictive factor for unfavorable overall survival (hazard ratio = 2.060, 95% confidence interval = [1.256, 3.381], p = 0.004) in a cohort of 181 hepatitis-B-related, sorafenib-treated HCC patients (concomitant tenofovir versus entecavir treatment; p = 0.003). In conclusion, by conducting a glycoproteome-wide search for GalNAc-T14 substrates, we unexpectedly found that tenofovir was a major negative regulator of GalNAc-T14 substrates and an unfavorable anti-hepatitis B drug in HCC patients receiving sorafenib.

Quantification of adenosine Mono-, Di- and triphosphate from royal jelly using liquid chromatography - Tandem mass spectrometry.

Nucleotides are composed of nitrogen bases, ribose units and phosphate groups. Adenine (Ade), adenosine monophosphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP) all play important roles in physiological metabolism. Royal jelly, a secretion produced by worker bees, contains a variety of natural ingredients and several studies have shown that royal jelly can serve as a source of nutrition for humans. In this study, a rapid and effective LC/ MS method coupled with pre-processing methods was developed and validated for the accurate quantification of Ade, AMP, ADP and ATP in royal jelly. To achieve the best extraction efficiency, two pretreatment methods, namely, solid-phase extraction (SPE) and dispersive solid-phase extraction (dSPE), were developed and investigated. Silica-based cyanopropyl (CN) liquid chromatography was employed using pH programming with a quaternary mobile phase system for the analyses. The total LC/MS run time was less than 12 min with a constant flow rate of 0.25 mL/min. The linear range were 2.5-1000 ng/mL with a correlation coefficient r = 0.9995. The limit of detection (LOD) of Ade, AMP, ADP and ATP was 1, 1, 2.5 and 5 ng/mL; the limit of quantitation (LOQ) was 2.5, 2.5, 5 and 10 ng/mL, respectively. Precision (RSD% <10.5%) and accuracy (recovery 81.3-118.4%) were satisfactory for both two pre-processing methods. Nucleotides were successfully quantified from 2-day and 3-day royal jelly samples, with concentrations within 6.2-2126.0 mg/kg.

Differential dynamics of hepatic protein expressions with long-term cultivated hepatitis C virus infection.

BACKGROUND: The liver maintains blood chemical homeostasis by active uptake and secretion through endocytosis, exocytosis, and intracellular trafficking between the plasma and intracellular membranes. Hepatitis C virus (HCV) infection affects the host membrane architecture and might thus impair the regulation of the cellular transportation machinery. Additionally, the hepatic expressions of differential protein dynamics with long-term HCV infection remain fully recover. METHODS: In this study, comparative proteomic analysis was performed in HCV-infected and mock-control Huh7 cells according to the viral dynamics of exponential, plateau, declined, and silencing phases at the acute stage, and the chronic stage. The proteins with <0.8-fold and ≥1.25-fold changes in expression were analyzed using functional pathway clustering prediction. RESULTS: The combined experimental repetitions identified full-spectrum cellular proteins in each of 5 sample sets from acute exponential, plateau, declined, and silencing phases, and the chronic stage. The clustering results revealed that HCV infection might differentiate regulatory pathways involving extracellular exosome, cadherin, melanosome, and RNA binding. Overall host proteins in HCV-infected cells exhibited kinetic pattern 1, in which cellular expression was downregulated from the acute exponential to plateau phases, reached a nadir, and was then elevated at the chronic stage. The proteins involved in the membrane-budding pathway exhibited kinetic pattern 2, in which their expressions were distinctly downregulated at the chronic stage. CONCLUSION: The current comparative proteomics revealed the differential regulatory effects of HCV infection on host intracellular transport functional pathways, which might contribute to the pathogenic mechanisms of HCV in hepatocytes that sustain long-term infection.

The possible interaction of CDA14 and protein elongation factor 1alpha.

The CDA14, a 45kD protein, is currently annotated as PTX1-like protein or ERGIC 32. Over expressing CDA14 can slow PC11 cell proliferation rate. In HepG2 cells, it had been demonstrated that CDA14 is involved in protein transportation. The knowledge about the protein is very limited and not clarified. The CDA14 and its homologous proteins form a family and are restricted to eukaryotes. In the family, there are no homologous sequences with resolved three-dimensional structure and their functions are difficult to predict. Transcriptional expression of CDA14 in three hepatoma cell lines, Hu7, HCC and HepG2, was lower than normal liver tissue and liver carcinoma tissue. In this study, functional proteomic techniques were utilized in searching the interacting counterpart of CDA14. Several proteins involved in protein translation and folding were selectively precipitated with CDA14 and identified mass spectrometry. Interaction of CDA14 and elongation factor 1alpha was confirmed by Western blotting and confocal microscopy. Elongation factor 1alpha is a multiple function protein and involved in several biological mechanisms, including protein synthesis, cell proliferation, apoptosis and tumorigensis. Over-expression of CDA14 down regulated the proliferation of HepG2 cells. These results suggest that CDA14 participated in the elongation factor 1alpha regulated mechanisms.