Additional file 1: of Estimated divergence times of Hirsutella (asexual morphs) in Ophiocordyceps provides insight into evolution of phialide structure

Table S1. GenBank accession numbers for sequences used in the phylogenetic analysis of Hirsutella (asexual morph). Table S2. Morphological comparison among Ophiocordyceps retorta and its related species. (DOC 86 kb

Citric Acid-Assisted Two-Step Enrichment with TiO₂ Enhances the Separation of Multi- and Monophosphorylated Peptides and Increases Phosphoprotein Profiling

Phosphopeptide enrichment is essential for large-scale phosphoprotein profiling. Titanium dioxide (TiO₂) is widely used in phosphopeptide enrichment, but it is limited in the isolation of multiphosphorylated peptides due to their strong binding. In this study, we found that citric acid greatly affects the binding of mono- and multiphosphopeptides with TiO₂, which can be used for stepwise phosphopeptide separation coupled with mass spectrum (MS) identification. We first loaded approximately 1 mg of peptide mixture of HeLa cell digests onto TiO₂ beads in highly concentrated citric acid (1 M). Then the flow-through fraction was diluted to ensure low concentration of citric acid (50 mM) and followed by loading onto another aliquot of TiO₂ beads. The two eluted fractions were subjected to nanoLC–MS/MS analysis. We identified 1,500 phosphorylated peptides, of which 69% were multiphosphorylated after the first enrichment. After the second enrichment, 2,167 phosphopeptides, of which 92% were monophosphorylated, were identified. In total, we successfully identified 3,136 unique phosphopeptides containing 3,973 phosphosites utilizing this strategy. Finally, more than 37% of the total phosphopeptides and 2.6-fold more of the multiphosphorylated peptides were identified as compared to the frequently used DHB/TiO₂ enrichment strategy. Combining SCX with CATSET, we identified 14,783 phosphopeptides and 15,713 phosphosites, of which 3,678 were unrecorded in PhosphoSitePlus database. This two-step separation procedure for sequentially enriching multi- and monophosphorylated peptides by using citric acid is advantageous in multiphosphorylated peptide separation, as well as for more comprehensive phosphoprotein profiling

Citric Acid-Assisted Two-Step Enrichment with TiO₂ Enhances the Separation of Multi- and Monophosphorylated Peptides and Increases Phosphoprotein Profiling

Phosphopeptide enrichment is essential for large-scale phosphoprotein profiling. Titanium dioxide (TiO₂) is widely used in phosphopeptide enrichment, but it is limited in the isolation of multiphosphorylated peptides due to their strong binding. In this study, we found that citric acid greatly affects the binding of mono- and multiphosphopeptides with TiO₂, which can be used for stepwise phosphopeptide separation coupled with mass spectrum (MS) identification. We first loaded approximately 1 mg of peptide mixture of HeLa cell digests onto TiO₂ beads in highly concentrated citric acid (1 M). Then the flow-through fraction was diluted to ensure low concentration of citric acid (50 mM) and followed by loading onto another aliquot of TiO₂ beads. The two eluted fractions were subjected to nanoLC–MS/MS analysis. We identified 1,500 phosphorylated peptides, of which 69% were multiphosphorylated after the first enrichment. After the second enrichment, 2,167 phosphopeptides, of which 92% were monophosphorylated, were identified. In total, we successfully identified 3,136 unique phosphopeptides containing 3,973 phosphosites utilizing this strategy. Finally, more than 37% of the total phosphopeptides and 2.6-fold more of the multiphosphorylated peptides were identified as compared to the frequently used DHB/TiO₂ enrichment strategy. Combining SCX with CATSET, we identified 14,783 phosphopeptides and 15,713 phosphosites, of which 3,678 were unrecorded in PhosphoSitePlus database. This two-step separation procedure for sequentially enriching multi- and monophosphorylated peptides by using citric acid is advantageous in multiphosphorylated peptide separation, as well as for more comprehensive phosphoprotein profiling

The immuohistochemical staining of atherosclerosis in the 3 groups.

<p>Legend: Magnifications: ×400. MMP-9: matrix metalloproteinase-9; LOX-1: lectin-like oxidized low density lipoprotein receptor-1. Representative pictures of MMP-9 and LOX-1 expressions immuohistochemical staining in the aortic atherosclerotic plaque of the three groups.</p

The IMT of the three groups in different weeks (mean [SD], mm).

<p>IMT: intima-media thickness.</p>#<p>: <i>P</i><0.05,</p><p>*: <i>P</i><0.01,</p>a<p>: compared with 0 week,</p>b<p>: compared with week 12;</p>c<p>: compared with group 1,</p>d<p>: compared with group 2.</p

The fluctuations of serum lipids level in the three groups.

<p>The fluctuations of serum lipids level in the three groups.</p

SI values in the three groups (mean [SD]).

<p>LDL-C: low-density lipoprotein cholesterol, SI: smoothness index, TC: total cholesterol.</p><p>*: <i>P</i><0.01,</p>c<p>: compared with group 1,</p>d<p>: compared with group 2.</p

The H&E staining of atherosclerosis in the three groups.

<p>Legend: Magnifications: ×200(A–C2), ×400(C3). A: Normal aorta intima in group 1. B1–B2: Atherosclerotic plaque in group 2. The endothelial cells shed, numerous foam cells and cholesterol crystal (B1); cell necrosis and calcium deposition (B2). C1–C3: Atherosclerotic plaque in group 3. plaques with thin fibrous caps and big lipid cores (C1), the discontinuous fiber cap (C2), inflammatory cells (C3).</p

The intima with Oil Red O staining of atherosclerosis in the three groups.

<p>The intima with Oil Red O staining of atherosclerosis in the three groups.</p

Body weight in the three groups (mean [SD]).

#<p>: <i>P</i><0.05,</p><p>*: <i>P</i><0.01,</p>a<p>: compared with 0 week,</p>b<p>: compared with week 12.</p