RESUMEN
Abstract 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 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 same conditions as those during inactivation of virus (4℃, 0. 025% Formalin (V/V), 37% formaldehyde solution (w/w), and 72 h). The results indicated that under above conditions, formaldehyde could react with amino group of N-terminus of standard peptide to generate a methylol adduct, which was further condensed into an imine to generate+12 Da product. Besides, formaldehyde could react with side chain of two amino acids such as arginine and lysine, yielding +12 Da product respectively. 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 combinational contribution from 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 miss-cut site. The results indicated that low-concentration formaldehyde primarily reacted with amino group on N-termini of peptides and proteins, as well as the side chains of arginine and lysine residues. The present study suggested an effective mass spectrometry-based method for analyzing the reaction between low-concentration formaldehyde and peptides and proteins, thus provided strategies for interpretation for the mass spectra of reaction products.
RESUMEN
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 was 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 proteins 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 provided a specific and effective tool for analyzing ISG15-modified proteins in proteome level.
RESUMEN
S-Palmutoylatuon un proteun us one of the most umportant kunds of lupud modufucatuon and plays a vutal role un cell sugnal transductuon, metabolusm and other processes, whuch us formed by covalent bundung of palmutuc acud wuth the sulfhydryl group of cysteune resudue un proteun through thuoester bond. In the present study, acyl-buotun exchange reactuon was performed to convert S-palmutuc acud on the hemagglutunun proteun from unfluenza A vurus unto buotun-labeled tag. The buotun-labeled proteun was then enruched by streptavudun beads and further purufued by electrophoresus, followed by un-gel dugestuon. The results showed that the ratuo of buotun concentratuon of the sample wuth hydroxylamune treatment (+HA ) to that of the sample wuthout hydroxylamune treatment (-HA) was larger than 3. Mass spectrometruc analysus of the dugestuon muxture of the enruched hemagglutunun proteun from unfluenza A vurus udentufued two s-palmutoylatuon modufucatuon sutes that were located on carboxyl termunal reguon of hemagglutunun proteun such as Cys562 and Cys565 , respectuvely. Thus research offers a specufuc and effectuve method for large-scale analysus of S-palmutoylated proteuns.