The cellular processes underpinning life are orchestrated by proteins and their interactions. The associated structural and dynamic heterogeneity, despite being key to function, poses a fundamental challenge to existing analytical and structural methodologies. We used interferometric scattering microscopy to quantify the mass of single biomolecules in solution with 2% sequence mass accuracy, up to 19-kilodalton resolution, and 1-kilodalton precision. We resolved oligomeric distributions at high dynamic range, detected small-molecule binding, and mass-imaged proteins with associated lipids and sugars. These capabilities enabled us to characterize the molecular dynamics of processes as diverse as glycoprotein cross-linking, amyloidogenic protein aggregation, and actin polymerization. Interferometric scattering mass spectrometry allows spatiotemporally resolved measurement of a broad range of biomolecular interactions, one molecule at a time.

Transcription factor p53 induces cell cycle arrest and apoptosis in response to DNA damage 1 and cellular stress, 2 thereby playing a critical role in protecting cells from malignant transformation. The E3 ubiquitin ligase hDM2 controls p53 levels through a direct binding interaction that neutralizes p53 transactivation activity, exports nuclear p53, and targets it for degradation via the ubiquitylation-proteasomal pathway. 3,4 Loss of p53 activity, either by deletion, mutation, or hDM2 overexpression, is the most common defect in human cancer. 5 Tumors with preserved expression of wild type p53 are rendered vulnerable by pharmacologic approaches that stabilize native p53. In this context, hDM2 targeting has emerged as a validated approach to restore p53 activity and resensitize cancer cells to apoptosis in vitro and in vivo. 6 Supporting Information Available: Complete ref 16h, peptide characterization, experimentals, and supplementary figures. This material is available free of charge via the Internet at http://pubs.acs.org.

Immune-based self-recognition and failure to modulate this response are believed to contribute to the debilitating autoimmune pathology observed in multiple sclerosis (MS). Studies from its murine model, experimental autoimmune encephalomyelitis (EAE), have shown that neuroantigen-specific CD4+ T cells are capable of inducing disease, while their immune sibling, the CD8+ T cells, have largely been ignored. To understand their role in autoimmune demyelination, we first confirmed that, similar to our observations in human MS, there is robust induction of neuroantigen-reactive CD8+ T cells in several models, including MOG 35-55 /CFA-induced EAE. However, MOG 35-55 -specific CD8+ T-cells, when purified, were unable to adoptively transfer disease into naïve mice (in contrast to CD4+ T-cells). In fact, we observed that the transfer of these neuroantigen-specific CD8+ T cells was able to suppress the induction of EAE and to inhibit ongoing EAE. These regulatory CD8+ T cells produced IFN-γ and perforin and were able to kill MOG loaded CD4+ T-cells as well as CD4-depleted APC, suggesting a cytotoxic/suppressor mechanism. Inhibition of EAE was associated with both the modulation of APC function as well as decreased MOG-specific CD4+ T cell responses. Our studies reveal a novel and unexpected immune regulatory function for neuroantigen-specific CD8+ T cells and have interesting biologic and therapeutic implications.