S-palmitoylation in protein is one of the most important kinds of lipid modification and plays a vital role in cell signal transduction, metabolism and other processes. S-palmitoylation is formed by covalently binding of palmitic acid with sulfhydryl group of cysteine residue in protein through thioester bond. In this study, acyl-biotin exchange reaction was firstly used to convert S-palmitic acid on the hemagglutinin protein from influenza A virus into biotin-labeled tag. The biotin-labeled protein was then enriched by streptavidin-linked beads and further purified by electrophoresis, followed by in-gel digestion. The results showed that the ratio of biotin concentration of the sample with hydroxylamine treatment (+HA) to that of the sample without hydroxylamine treatment (−HA) was larger than 3. Mass spectrometric analysis of the digestion mixture of the enriched hemagglutinin protein from influenza A virus identified two S-palmitoylation modification sites that were located on carboxyl terminal region of hemagglutinin protein such as Cys562 and Cys565, respectively. This research offers a specific and effective method for large-scale analysis of S-palmitoylated proteins.The present study proposed a quality control parameter for acyl-biotin exchange reaction. Mass spectrometry, coupled with acyl-biotin exchange and affinity enrichment, was used to identify two S-palmitoylation modification sites in hemagglutinin protein from influenza A virus.

Interferon stimulated gene 15 kDa protein (ISG15) is the first ubiquitin-like protein identified, which plays vital roles in a variety of fields including viral infection and immunological regulation. In this study, liquid chromatography-tandem mass spectrometry was used to analyze ISG15-modified proteins in A549 cells in response to infection by influenza virus, which were enriched by immunoprecipitation. A total of 22 cellular host proteins were identified in A549 cells infected by influenza virus, including ubiquitin-like ISG15 protein, cyclin-T1, heat shock protein 71 kDa, caldesmon, eukaryotic translation initiation factor, and so on. Besides, non-structural protein (NS1) from influenza virus was also identified. Among the 22 host proteins identified, 6 were also identified in the control non-infected A549 cells, including annexin A1, fructose-bisphosphate aldolase A, ATP synthase subunit g, enolase, actin, and tubulin. Bioinformatics analysis revealed that the identified ISG15-modified host proteins induced by influenza virus infection could be classified into 9 protein classes: chaperone, oxidoreductase, enzyme modulator, transferase, nucleic acid binding, transcription factor, kinase, cytoskeletal protein, and structural protein. This study provides a specific and effective tool for analyzing ISG15-modified proteins at proteome level.Cellular proteins modified by ISG15 are enriched by immunoprecipitation, and then analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Formaldehyde has been widely employed to immobilize clinical tissue specimens, inactivate toxins and viruses in biomedical fields. Formaldehyde can react with active groups in bio-molecules such as proteins, resulting in protein cross-linking, inactivation and immobilization. By using several standard peptides and tryptic peptides from the matrix protein of influenza virus as experimental models, we studied the chemical modifications of peptides and proteins with formaldehyde by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and nano-electrospray quadruple time-of-flight tandem mass spectrometry. The reaction between formaldehyde and peptides was performed under the conditions consistent with those during inactivation of virus (4 °C, 0.025% Formalin (37% (w/w) formaldehyde solution), and 72 h). The results indicated that under the above conditions, formaldehyde reacted with amino groups of N-termini of standard peptides to generate methylol adducts, which was further condensed into imines to generate +12 Da products. Furthermore, formaldehyde could react with side chain of two amino acids such as arginine and lysine, yielding +12 Da product each. The analysis of the reaction between formaldehyde and tryptic peptides from matrix protein of influenza virus showed that +24 Da products could be detected in most peptides due to the combinational contribution of N-terminus of peptide (+12 Da) and side chain of C-terminal arginine or lysine (+12 Da). Moreover, a +36 Da product was detected for a peptide with an internal miss-cut site. The results indicated that low-concentration formaldehyde primarily reacted with amino groups on N-termini of peptides and proteins, as well as the side chains of arginine and lysine residues. The present study provided an effective mass spectrometry-based method for analyzing the reaction between low-concentration formaldehyde and peptides and proteins, as well as strategies for interpretation for the mass spectra of reaction products.Mass spectrometry analysis reveals that low concentration formaldehyde can readily react with the free amino groups of both N-terminal and side chain of protein to generate a methylol adduct, which is then condensed into an imine (+12Da) by losing a molecule of water.

In order to identify potential protein targets involved in colorectal cancer(CRC), we used a liquid chromatography coupled with mass spectrometry(LC-MS)/MS-based proteomics approach to characterize global protein expression patterns in malignant tissues and their adjacent healthy tissues from Dukes C stage CRC patients. A total number of 34 differentially expressed proteins were detected and identified by LC-MS/MS and database searching, which are supposed to be relevant to progression of colorectal tumor. Among these proteins, nucleophosmin 1(NPM1) was found to be remarkably up-regulated in colorectal carcinoma tissues, as compared with that in their normal counterparts. The results presented here could provide clues to elucidate the pathological significance of NPM1 in regulation of carcinogenesis of Dukes C stage colorectal tumors.

Protein sample preparation is a vital issue for proteomics research. Due to different physical and chemical properties of reagents used for sample preparation, the ability of different reagents to disrupt cell or tissue, as well as to solubilize a variety of proteins, is very different. In the present study, three sample preparation methods widely used in proteomics researches (Triton X-100 method, urea method and TRIzol method) were compared by using mass spectrometric method in analyzing proteins isolated from cultured 293T cells, followed by bioinformatics analyses. The results indicated that the number of the identified proteins extracted by Triton X-100 method was almost the same as that by urea method, whereas the number of the identified proteins extracted by TRIzol method was approximately 8 percent smaller than those by either Triton X-100 or urea method. A large difference was found among the protein categories identified by three extraction methods, and only 32 percent proteins were identified from samples by all three methods. The profiles of proteins prepared by three methods were compared and further analyzed using functional classification software. This study provides a rapid, effective and comprehensive tool for evaluating the sample preparation methods for proteomics study.Three sample preparation methods (Triton X-100 method, urea method and TRIzol method) were evaluated. The profiles of proteins prepared by three methods were compared and further analyzed using bioinformatics tools. This study provides a rapid, effective and comprehensive tool for evaluating the sample preparation methods for proteomics study.

In the present study, 14-3-3ε protein was over-expressed in HEK293 cells, which was genetically attached with 6 × His tag. The protein was purified and enriched by nickel-charged resin and gel electrophoresis, and then was subject to tryptic digestion. The phosphorylated peptide fragments within 14-3-3ε protein were enriched by TiO2 affinity chromatography, followed by mass spectrometry analysis. Totally twelve phosphorylation sites along the primary structure of 14-3-3ε protein were identified, most of which had not been reported previously. The results indicated that bio-mass spectrometry coupled with manual interpretation was quite feasible to identify the phosphorylation modification in 14-3-3ε protein over-expressed in HEK293 cells.

In this study, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) coupled with mass spectrometry was used to perform a comprehensive proteomics analysis of the purified influenza virus particles propagated from embryonated chicken eggs. In addition to the virus-encoded proteins, 12 proteins originated from embryonated chicken eggs were identified in the purified virus particles, such as annexin A2, peptidyl-prolyl cis-trans isomerase B, peroxiredoxin-1, phosphoglycerate kinase, and pyruvate kinase muscle isoenzyme. Two kinds of cytoskeletal proteins, tubulin b-3 and actin, were also detected.

Oxygen exchange may occur at carboxyl groups catalyzed by acid. The reaction, however, takes at least several days at room temperature. The long-time exchanging reaction often prevents its application from protein analysis. In this study, an 18O-labeling method utilizing microwave-assisted acid hydrolysis was developed. After being dissolved in 16O/18O (1:1) water containing 2.5% formic acid, protein samples were exposed to microwave irradiation. LC-MS/MS analysis of the resulted peptide mixtures indicated that oxygen in the carboxyl groups from glutamic acid, aspartic acid, and the C-terminal residues could be efficiently exchanged with 18O within less than 15 min. The rate of back exchange was so slow that no detectable back exchange could be found during the HPLC run.

•17-Hydroxy-jolkinolide A (HJA) is an ent-abietane diterpenoid, isolated from the dried root of Euphorbia fischeriana.•HJA inhibited RANKL-induced formation of osteoclasts from BMMs.•HJA prevented formation of bone resorption pits by mature osteoclasts.•HJA suppressed activation of NF-κB and MAPK signaling.Osteoclasts (OC) are bone-specific multinucleated giant cells (MNCs) derived from the monocyte/macrophage hematopoietic lineage cells. Inhibiting osteoclast formation is considered as an effective therapeutic approach for the treatment of the pathological bone loss. In this study, we investigated effects of 17-hydroxy-jolkinolide A (HJA), an ent-abietane diterpenoid isolated from the dried root of Euphorbia fischeriana, on osteoclastogenesis induced by RANKL. The results showed that HJA significantly inhibited RANKL-induced osteoclast formation from primary bone marrow macrophages (BMMs). HJA also prevented bone resorption by mature osteoclasts in a dose-dependent manner. In addition, the expression of osteoclastic marker genes, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K (Cts K) and MMP-9, was significantly inhibited by HJA. Furthermore, HJA also significantly inhibited RANKL-induced activation of NF-κB and phosphorylation of MAPK. Our results indicate that HJA has an inhibitory role in the bone loss by preventing osteoclast formation as well as its bone resorptive activity. Therefore, HJA may be useful as a therapeutic reagent for bone loss-associated diseases.