Perlman, D. H. | bioRxiv Science

bioRxiv2018

Automated sample preparation for high-throughput single-cell proteomics

A major limitation to applying quantitative LC-MS/MS proteomics to small samples, such as single cells, are the losses incured during sample cleanup. To relieve this limitation, we developed a Minimal ProteOmic sample Preparation (mPOP) method for culture-grown mammalian cells. mPOP obviates cleanup and thus eliminates cleanup-related losses while expediting sample preparation and simplifying its automation. Bulk SILAC samples processed by mPOP or by conventional urea-based methods indicated that mPOP results in complete cell lysis and accurate relative quantification. We integrated mPOP lysis with the Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS) sample preparation, and benchmarked the quantification of such samples on a Q-exactive instrument. The results demonstrate low noise and high technical reproducibility. Then, we FACS sorted single U-937, HEK-293, and mouse ES cells into 96-well plates and analyzed them by automated mPOP and SCoPE-MS. The quantified proteins enabled separating the single cells by cell-type and cell-division-cycle phase.

systems biology↗

bioRxiv2018

Protein nitration is a physiological regulator of cardiac lactate dehydrogenase active site loop mobility and activity

Protein tyrosine nitration is a hallmark of oxidative stress related disease states, commonly detected as anti-nitrotyrosine immunoreactivity. The precise reactive oxygen sources, mechanisms of nitration as well as the modified target proteins and functional consequences, however, remain often unclear. Here we explore protein tyrosine nitration under basal conditions and find surprisingly physiologically nitrated proteins. Upon purifying a prominent physiologically nitrotyrosine immunopositive in hearts from mouse, rat and pig, we identify it as lactate dehydrogenase (LDH). Mechanistically, LDHs degree of basal nitration depended on two canonical sources, NO synthase (NOS) and myeloperoxidase (MPO), respectively. When validating the nitrated amino acid by MALDI-TOF mass spectrometry, we, surprisingly, located LDH nitration not to a tyrosine but the C-terminal tryptophan, Trp324. Molecular dynamics simulations suggested that Trp324 nitration restricts the interaction of the active site loop with the C-terminal -helix essential for activity. This prediction was confirmed by enzyme kinetics revealing an apparent lower Vmax of nitrated LDH, although yet unidentified concurrent oxidative modifications may contribute. Protein nitration is, thus, not a by definition disease marker but reflects also physiological signaling by eNOS/NO, MPO/nitrite and possibly other pathways. The commonly used assay of anti-nitrotyrosine immunoreactivity is apparently cross-reactive to nitrotryptophan requiring a reevaluation of the protein nitration literature. In the case of LDH, nitration of Trp324 is aggravated under cardiac metabolic stress conditions and functionally limits maximal enzyme activity. Trp324-nitrated LDH may serve both as a previously not recognized disease biomarker and possibly mechanistic lead to understand the metabolic changes under these conditions.

biochemistry↗