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Sui, J.-H.

Publications and source records attributed to Sui, J.-H..

2 recordsLinked to original sources
bioRxiv2024

Do-it-yourself de novo antibody sequencing workflow that achieves complete accuracy of the variable regions

Antibodies are widely used as research tools or therapeutic agents. Knowing the sequences of the variable regions of an antibody--both the heavy chain and the light chain--is a prerequisite for the production of recombinant antibodies. Mass spectrometry-based de novo sequencing is a frequently used, and sometimes the only approach to gaining this information. Here, we describe a workflow that enables accurate sequence determination of monoclonal antibodies based on mass spectrometry data and freely available software tools. This workflow, which we developed using a homemade anti-FLAG monoclonal antibody as a reference sample, achieved 100% accuracy of the variable regions with clear distinction between leucine (L) and isoleucine (I). Using this workflow, we successfully decoded a monoclonal anti-HA antibody, for which we had no prior knowledge of its sequence. Based on the de novo sequencing result, we generated a recombinant anti-HA antibody, and demonstrated that it has the same specificity, sensitivity, and affinity as the commercial antibody. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/630060v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@180c502org.highwire.dtl.DTLVardef@1064399org.highwire.dtl.DTLVardef@4005d3org.highwire.dtl.DTLVardef@13e65ed_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗
bioRxiv2024

Global analysis of protein and small-molecule substrates of ubiquitin-like proteins (UBLs)

AbstractUbiquitin-like proteins (UBLs) constitute a family of evolutionarily conserved proteins that share similarities with ubiquitin in 3D structures and modification mechanisms. For most UBLs including Small-Ubiquitin-like Modifiers (SUMO), their modification sites on substrate proteins cannot be identified using the mass spectrometry-based method that has been successful for identifying ubiquitination sites, unless a UBL protein is mutated accordingly. To identify UBL modification sites without having to mutate UBL, we have developed a dedicated search engine pLink-UBL on the basis of pLink, a software tool for identification of cross-linked peptide pairs. pLink-UBL exhibited superior precision, sensitivity, and speed than "make-do" search engines such as MaxQuant, pFind, and pLink. For example, compared to MaxQuant, pLink-UBL increased the number of identified SUMOylation sites by 50 [~] 300% from the same datasets. Additionally, we present a method for identifying small-molecule modifications of UBLs. This method involves antibody enrichment of a UBL C-terminal peptide following enrichment of a UBL protein, followed by LC-MS/MS analysis and a pFind 3 blind search to identify unexpected modifications. Using this method, we have discovered non-protein substrates of SUMO, of which spermidine is the major one for fission yeast SUMO Pmt3. Spermidine can be conjugated to the C-terminal carboxylate group of Pmt3 through its N1 or also likely, N8 amino group in the presence of SUMO E1, E2, and ATP. Pmt3-spermidine conjugation does not require E3 and can be reversed by SUMO isopeptidase Ulp1. SUMO-spermidine conjugation is present in mice and humans. Also, spermidine can be conjugated to ubiquitin in vitro by E1 and E2 in the presence of ATP. The above observations suggest that spermidine may be a common small molecule substrate of SUMO and possibly ubiquitin across eukaryotic species. HighlightsO_LIA specialized software pLink-UBL enables precise identification of UBL modification sites on protein substrates. C_LIO_LIpFind 3 blind search enables identification of unexpected small-molecule substrates of a UBL protein. C_LIO_LISpermidine is a small molecule substrate of fission yeast SUMO Pmt3 as well as mammalian SUMO proteins. C_LIO_LIThe C-terminal carboxyl group of Pmt3 can be attached to the N1 or likely also the N8 amino group of spermidine in the presence of E1, E2, and ATP, and can be detached by SUMO isopeptidase. C_LI

biochemistry↗