HR-MS of the LLEIEDQLEEAAVFPGK peptide showing the modified and unmodified states for <i>z</i> = 2 and <i>z</i> = 3.

<p>Numbers at the top are the theoretical and experimentally observed masses of the peptide.</p

Rhamnose Links Moonlighting Proteins to Membrane Phospholipid in Mycoplasmas - Fig 2

<p>GC/MS of a rhamnose standard (panel A), IP-purified membrane enolase isolated from a SDS-PAGE gel (panel B), and the same material as analyzed in panel B after subsequent treatment with phospholipase D (panel C).</p

SDS-PAGE analysis of <i>M</i>. <i>pulmonis</i>.

<p>Panel A, Coomassie-stained gel of supernatants obtained by treatment of whole cells with phospholipase D (PLD) or heat-killed enzyme (HK-PLD). Proteins were eluted from the gel in wide swaths that were analyzed by ESI-TOF MS for protein identification (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162505#pone.0162505.t001" target="_blank">Table 1</a>). Panel B, membrane proteins were analyzed on gels stained with Coomassie, Pro-Q Emerald (Glycostain), Pro-Q Diamond (Phosphostain), Western blot reacted with antibody to enolase, and a Coomassie-stained gel of IP-purified enolase. Panel C, IP-purified enolase from membrane and cytoplasmic fractions were analyzed on gels stained for total protein (Coomassie), glycoprotein, or phosphoprotein. In all panels numbers refer to the masses of protein standards, and arrows refer to the expected location of enolase.</p

HPLC plot of the relative abundance of the LLEIEDQLEEAAVFPGK peptide of enolase.

<p>The inset is a plot of another peptide from enolase that lacks lipid modification.</p

MS/MS of the LLEIEDQLEEAAVFPGK peptide showing modification at Gln437.

<p>Inset A. Model for the 128.05 m/<i>z</i> residue. Inset B. Model of the PTM associated with membrane enolase. The model is based on phosphatidylcholine because this lipid is a main substrate for phospholipase D and is a major lipid for mycoplasmas grown in serum [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162505#pone.0162505.ref042" target="_blank">42</a>].</p

LC MS/MS-CID showing hexosylation at Asn335 of the peptide STLEYTINNSQELQn₃₃₅ILKQTYEEFTK of MYPU_3200.

<p>The assigned b and y ions are shown in blue and red, respectively. Glycosylation of N, Q, T, S, and Y glycosites is absent in this spectrum as illustrated. The PEAKS peptide score (-10lgP) for this spectrum was 60. The charge state of the parental ion was <i>z</i> = 3.</p

The effect of substrate on glycoconjugate synthesis by <i>M</i>. <i>pulmonis</i> and <i>M</i>. <i>pneumoniae</i>.

<p>Panels A and B show the relative abundance of glucose or xylose, respectively, linked to protein as determined by GC. The values represent averages of the areas under the curves from 3 replicates of gas chromatograms that were converted to μg of sugar per mg of protein. The colors of the bars representing the different substrates used to supplement the medium are shown on the right. Plus or minus standard error bars are shown and the asterisks indicate a significant difference between a sample and control.</p

Hexosylation of the peptide GTKDFLPIELQSLEVSK of MYPU_3230.

<p>Orbitrap MS showing the doubly and triply charged ions. The 81.0262 shift for <i>z</i> = 2 between the non-glycosylated and glycosylated peptides equates to a mass shift of 162.0524 Da, which corresponds to the addition of hexose (162.0528 Da) with a mass accuracy of 0.0004 Da. The 54.0169 shift for <i>z</i> = 3 between non-glycosylated and glycosylated forms equates to a mass shift of 162.0507 Da, which corresponds to hexosylation with a mass accuracy of 0.0021 Da. Monoisotopic values for the calculated theoretical and experimental masses of the peptide are given in bold. The images presented were obtained from an LC peak of MS scans and are expanded to show the charge states of each form.</p

Hexosylation of the peptide STLEYTINNSQELQNILKQTYEEFTK of MYPU_3200.

<p>Orbitrap MS showing the doubly and triply charged ions. The monoisotopic mass of the doubly charged species at 1648.8077 is consistent with the hexosylated peptide at <i>z</i> = 2 with a mass accuracy of 0.0012 Da. The 54.0175 shift for <i>z</i> = 3 between non-glycosylated and hexose forms equates to a mass shift of 162.0525 Da with a mass accuracy of 0.0003 Da. Monoisotopic values for the calculated theoretical and experimental masses of the peptide are given in bold. The images presented were obtained from an LC peak of MS scans and are expanded to show the charge states of each form.</p

Glycosites identified in this study.

<p>^a Accession numbers for MYPU_3200, MYPU_3230, MYPU_3460, and MARTH_403 are CAC13493, CAC13496, CAC13519, and YP_001999972, respectively.</p><p>Glycosites identified in this study.</p