MOESM1 of Current applications of antibody microarrays

Additional file 1. Detailed information about the home-made and commercially available antibody microarrays

Global Identification of Prokaryotic Glycoproteins Based on an <em>Escherichia coli</em> Proteome Microarray

<div><p>Glycosylation is one of the most abundant protein posttranslational modifications. Protein glycosylation plays important roles not only in eukaryotes but also in prokaryotes. To further understand the roles of protein glycosylation in prokaryotes, we developed a lectin binding assay to screen glycoproteins on an <em>Escherichia coli</em> proteome microarray containing 4,256 affinity-purified <em>E.coli</em> proteins. Twenty-three <em>E.coli</em> proteins that bound Wheat-Germ Agglutinin (WGA) were identified. PANTHER protein classification analysis showed that these glycoprotein candidates were highly enriched in metabolic process and catalytic activity classes. One sub-network centered on deoxyribonuclease I (sbcB) was identified. Bioinformatics analysis suggests that prokaryotic protein glycosylation may play roles in nucleotide and nucleic acid metabolism. Fifteen of the 23 glycoprotein candidates were validated by lectin (WGA) staining, thereby increasing the number of validated <em>E. coli</em> glycoproteins from 3 to 18. By cataloguing glycoproteins in <em>E.coli</em>, our study greatly extends our understanding of protein glycosylation in prokaryotes.</p> </div

Glycoproteins identified by <i>E. coli</i> proteome microarrays and on-chip lectin (WGA) competition assays.

<p>Four representative novel glycoprotein candidates are shown. SNRs in the absence of glycan competitors and fold changes are given. The glycan competitor used for WGA probing was chitin hydrolysate.</p

Schematic diagram of glycan competition assays.

<p>Proteome arrays were probed with fluorescent dye conjugated lectin. <b>Left track</b>; One proteome chip was incubated with lectins, then washed to remove free lectins and some weak, non-specific interactions. Stronger non-specific interactions still occurred. <b>Right track</b>; A second proteome chip was incubated with lectins in the presence of excess amounts of glycan competitors to block glycan-dependent interactions. Glycoproteins were readily identified by comparing the signal intensities between the two microarrays, without and with glycan competitors.</p

The protein-protein interaction network of the 23 glycoprotein candidates.

<p>The network was mapped using STRING, and the confidence parameter was set as 0.15. Lines between proteins stand for possible interactions, and are color-coded based on the type of interaction.</p

Functional distribution of candidate proteins according to their (a) Biological process, (b) Molecular function and (c) Protein class.

<p>Categorizations were based on information provided by the online resource PANTHER classification system.</p

Validation of the novel glycoproteins by lectin blotting.

<p>A heavily glycosylated protein RNaseB was included as a positive control. The protein elution buffer used to purify the glycoproteins was used as a blank control. Red arrows indicate the target protein bands on the gel or the membrane. Results for the nine validated glycoproteins are shown. (<b>a–b</b>) Coomassie staining. (<b>c–d</b>) Western blotting using an anti-6xHis antibody. All of the glycoprotein candidates were 6xHis tagged at their C-terminals. (<b>e–f</b>) Lectin blotting using a biotinylated WGA followed by HRP-conjugated streptavidin.</p

Glycoproteins identified on <i>E.coli</i> proteome microarray by lectin probing.

<p>Glycoproteins identified on <i>E.coli</i> proteome microarray by lectin probing.</p

Global Profiling of Protein Lysine Malonylation in <i>Escherichia coli</i> Reveals Its Role in Energy Metabolism

Protein lysine malonylation is a recently identified post-translational modification (PTM), which is evolutionarily conserved from bacteria to mammals. Although analysis of lysine malonylome in mammalians suggested that this modification was related to energy metabolism, the substrates and biological roles of malonylation in prokaryotes are still poorly understood. In this study, we performed qualitative and quantitative analyses to globally identify lysine malonylation substrates in <i>Escherichia coli</i>. We identified 1745 malonylation sites in 594 proteins in <i>E. coli</i>, representing the first and largest malonylome data set in prokaryotes up to date. Bioinformatic analyses showed that lysine malonylation was significantly enriched in protein translation, energy metabolism pathways and fatty acid biosynthesis, implying the potential roles of protein malonylation in bacterial physiology. Quantitative proteomics by fatty acid synthase inhibition in both auxotrophic and prototrophic <i>E. coli</i> strains revealed that lysine malonylation is closely associated with <i>E. coli</i> fatty acid metabolism. Protein structural analysis and mutagenesis experiment suggested malonylation could impact enzymatic activity of citrate synthase, a key enzyme in citric acid (TCA) cycle. Further comparative analysis among lysine malonylome, succinylome and acetylome data showed that these three modifications could participate in some similar enriched metabolism pathways, but they could also possibly play distinct roles such as in fatty acid synthesis. These data expanded our knowledge of lysine malonylation in prokaryotes, providing a resource for functional study of lysine malonylation in bacteria

MOESM1 of Comparative analysis of human sperm glycocalyx from different freezability ejaculates by lectin microarray and identification of ABA as sperm freezability biomarker

Additional file 1. The characteristics of the sixty samples with different recovery rates