absolute-quantitation-inositol-pyrophosphates-capillary

Absolute Quantitation of Inositol Pyrophosphates by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry

Inositol pyrophosphates (PP-InsPs) are an important group of intracellular signaling molecules. Derived from inositol phosphates (InsPs), these molecules ...

University of Freiburg

A tunable chemoenzymatic strategy provides access to the entire class of magic spot nucleotides and modified analogues. The approach combines chemoselective bisphosphorylations using phosphoramidites with regioselective ribonuclease T2 cyclo-phosphate hydrolysis, leading to flexible and simple gram-scale operations.

Magic spot nucleotides: tunable target-specific chemoenzymatic synthesis.

T cell-independent (TI) B cell response is critical for the early protection against pathogen invasion. The regulation and activation of Bruton's tyrosine kinase (Btk) is known as a pivotal step of B cell antigen receptor (BCR) signaling in TI humoral immunity, as observed in patients with X-linked agammaglobulinemia (XLA) experiencing a high incidence of encapsulated bacterial infections. However, key questions remain as to whether a well-established canonical BCR signaling pathway is sufficient to regulate the activity of Btk. Here, we find that inositol hexakisphosphate (InsP) acts as a physiological regulator of Btk in BCR signaling. Absence of higher order inositol phosphates (InsPs), inositol polyphosphates, leads to an inability to mount immune response against TI antigens. Interestingly, the significance of InsP-mediated Btk regulation is more prominent in IgM plasma cells. Hence, the present study identifies higher order InsPs as principal components of B cell activation upon TI antigen stimulation and presents a mechanism for InsP-mediated regulation of the BCR signaling.

Inositol polyphosphates promote T cell-independent humoral immunity via the regulation of Bruton's tyrosine kinase.

The analysis of myo-inositol phosphates (InsPs) and myo-inositol pyrophosphates (PP-InsPs) is a daunting challenge due to the large number of possible isomers, the absence of a chromophore, the high charge density, the low abundance, and the instability of the esters and anhydrides. Given their importance in biology, an analytical approach to follow and understand this complex signaling hub is desirable. Here, capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry (ESI-MS) is implemented to analyze complex mixtures of InsPs and PP-InsPs with high sensitivity. Stable isotope labeled (SIL) internal standards allow for matrix-independent quantitative assignment. The method is validated in wild-type and knockout mammalian cell lines and in model organisms. SIL-CE-ESI-MS enables the accurate monitoring of InsPs and PP-InsPs arising from compartmentalized cellular synthesis pathways, by feeding cells with either [C]-myo-inositol or [C]-D-glucose. In doing so, we provide evidence for the existence of unknown inositol synthesis pathways in mammals, highlighting the potential of this method to dissect inositol phosphate metabolism and signalling.

Verena B. Eisenbeis

Institute of Organic Chemistry

University of Freiburg

Absolute Quantitation of Inositol Pyrophosphates by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry

Verena B. Eisenbeis

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Institute of Organic Chemistry

University of Freiburg

Absolute Quantitation of Inositol Pyrophosphates by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry

Institute of Organic Chemistry