Screening Of Affinity Peptide For Tyr Nitrated Peptides With Phage-displayed Peptide Library Technology

The tyrosine nitration of proteins play roles in the physiological and pathological processes of animals and plants.Among many human diseases,including diabetes,neurodegenerative diseases,cancer and so on,the level of tyrosine nitration of specific proteins increase.However,the abundance of nitrotyrosine in protein is very low,pre-separation and enrichment before the identification of nitrotyrosine sites is necessary.At present,the routine methods are mainly dependent on antibodies,but the commericial antibodies are expensive,and usually have preference for specific nitrotyrosine peptide sequences.In this study,phage screening technology was used to screen the affinity peptide of nitrotyrosine for enrichment of tyrosine nitrated peptides.First of all,nitroated GGGGY*GGG as the target,four rounds of screening were carried out in M13 phage random 12 peptide library,with reduced target concentration each round.Then the monoclonal of positive phage was determined by enzyme-linked immunosorbent assay.A highly repetitive phage clone with sequence TLWPFDLWLKTR was identified finally and named NT-1.Its repetition rate in positive phage is around 56.25 %.Next,the affinity between NT-1 and GGGGY*GGG was verified.When the concentration of GGGGY*GGG fixed on the matrix is 3.2 μM,the concentration of NT-1 in the solution were 6 μM,3 μM,1.5 μM,0.75μM,0.3 μM,30 n M,3 n M,0.3 n M respectively.The results of MALDI-TOF mass spectrometry show that the Signal/Noise(S/N)of enriched NT-1 are 1249.8,640.8,53.8,263.1,70.0,150.2,39.8,16.7,respectively.Furthermore,the specificity of NT-1 for GGGGY*GGG was tested in the presence of trypsin digested bovine serum albumin(BSA).When the concentration of GGGGY*GGG fixed on the substrate is 6.25μM,the concentration of free NT-1 is 2 μM,and the mass ratio of BSA hydrolysate to NT-1 is 100 and 200 fold,the S/N of NT-1 is 100.0,77.7,respectively;When the concentration of GGGGY*GGG fixed on the matrix is 62.5 μM,the concentration of free NT-1 is 220 n M,and the mass ratio of BSA hydrolysate to NT-1 is 500 and 1000 fold,NT-1 with S/N of7.2 and 6.8 can be identified in the enriched samples,respectively.Which shows that NT-1 has high affinity and specificity for GGGGY*GGG in semi complex samples.Then,in order to detect the affinity of NT-1 to different types of nitrotyrosine peptides,the sequence of 4547 reported nitrotyrosi ne peptides were analyzed and mainly grouped into 8 categories ac cording to neighboring amino acids of nitrotyrosine site: nitrotyrosin e coated by hydrophobicity,hydrophobicity-acid,acid-hydrophobicityacid,amide-hydrophobicity-alkaline,hydrophobicity-alkaline,alkaline,acid-serine,serine,respectively.According to the characteristics of the above motifs,we designed corresponding short epitope nitrotyrosine standard peptides: GGY*VA,GIY*EA,EY*LEE,NY*LK,AY*RQ,GY*KKL,DY*SG,SY*ST.Several glycine residues were added as a flexible arm,these are nitrotyrosine standard peptide used below.The short epitope nitrotyrosine standard peptides were mixed with trypsin digested BSA at different concentrations to explore the enrichment ability and selectivity of NT-1for these nitropeptides.In this system,the concentration of NT-1 fixed on the matrix is 1 μM and the concentration of free short epitope nitrotyrosine standard peptides is 800 n M.When the mass ratio of the trypsin digested BSA to nitrotyrosine standard peptides is 25 and 50 times,Seven or eight nitrotyrosine standard peptides were enriched on NT-1(GGGGAY*RQ and NY*LKGGGG have the same molecular weight,the same below).When BSA hydrolysate is increased to 200 times,Six or seven nitrotyrosine standard peptides were enriched on NT-1(Except for GGGGDY*SG).When BSA trypsin digestion is increased to 500 and1000 times,GGGGIY*EA and GGGGSY*ST can be enriched to NT-1.Due to the interferecence from degradation polymer of coupling matrix in acid elution,some peaks ranged from 500 to 1000 Da in MALDI-TOF MS were overlapped with partial target peaks of short epitope nitropeptides,especially from GGGGGY*VA.Furthermore,The NT-1 was enriched by nitrated standard short peptide fixed on matrix reversely and verified by mass spectrometry.In the system,eight short epitope standard peptides were fixed on the matrix(final concentration 6.25 μM),and the concentration of free NT-1 was 2 μM.The enriched NT-1 was identified by mass spectrometry after incubation with different concentration of BSA digest.When the mass ratio to the trypsin digest of BSA was 1:50 to 1:200,the NT-1 could be enriched to 8 short epitope standard peptides,confirmed the interaction between NT-1 and various short epitopes of nitrotyrosine peptides.Furthermore,NT-1 was applied to the enrichment of endogenou s nitrolated peptides from proteolytic products of biological samples.10 endogenous tyrosine nitropeptides,SSQESY*AHGTK,TLSSY*S TPK,DFMNEEY*SQEVR,QIDNPDY*KG,EDQTEY*LEER,EGVL Y*VGSK,LLLGGGSY*K,AGNTIHAILLY*R,LFEMAY*KK,SVSS SSY*R were synthesized.NT-1 coupled matrix(10 μM)was used t o enrich the free endogenous tyrosine nitropeptide(800 n M).The mass spectrometry results showed that NT-1 could enrich 8 endogen ous tyrosine nitropeptides without the presence of BSA digest.They are as follows: SVSSSSY*R,LLLGGGSY*K,EGVLY*VGSK,LF EMAY*KK,QIDNPDY*KG,AGNTIHAILLY*R,DFMNEEY*SQEV R,EDQTEY*LEER.When the ratio was 250,NT-1 could still be e nriched by 8 endogenous nitrolated peptides.When the ratio was 1000,NT-1 could be enriched by 7 endogenous nitrolated peptides.They are as follows: SVSSSSY*R,LLLGGGSY*K,EGVLY*VGSK,LFEMAY*KK,QIDNPDY*KG,AGNTIHAILLY*R,EDQTEY*LEE R.In summary,phage display technology could be an effective method to screen the peptide binding to nitrotyrosine.The NT-1 affinity peptide screened in this study has high affinity for various typical nitrotyrosine peptides.It is expected to be applied to the enrichment of endogenous tyrosine nitrolated peptides in more complex environments.