Low cell number proteomic analysis using in-cell protease digests reveals a robust signature for cell cycle state classification

Comprehensive proteome analysis of rare cell phenotypes remains a significant challenge. We report a method for low cell number MS-based proteomics using protease digestion of mildly formaldehyde-fixed cells in cellulo, which we call the “in-cell digest.” We combined this with averaged MS1 precursor library matching to quantitatively characterize proteomes from low cell numbers of human lymphoblasts. About 4500 proteins were detected from 2000 cells, and 2500 proteins were quantitated from 200 lymphoblasts. The ease of sample processing and high sensitivity makes this method exceptionally suited for the proteomic analysis of rare cell states, including immune cell subsets and cell cycle subphases. To demonstrate the method, we characterized the proteome changes across 16 cell cycle states (CCSs) isolated from an asynchronous TK6 cells, avoiding synchronization. States included late mitotic cells present at extremely low frequency. We identified 119 pseudoperiodic proteins that vary across the cell cycle. Clustering of the pseudoperiodic proteins showed abundance patterns consistent with “waves” of protein degradation in late S, at the G2&M border, midmitosis, and at mitotic exit. These clusters were distinguished by significant differences in predicted nuclear localization and interaction with the anaphase-promoting complex/cyclosome. The dataset also identifies putative anaphase-promoting complex/cyclosome substrates in mitosis and the temporal order in which they are targeted for degradation. We demonstrate that a protein signature made of these 119 high-confidence cell cycle–regulated proteins can be used to perform unbiased classification of proteomes into CCSs. We applied this signature to 296 proteomes that encompass a range of quantitation methods, cell types, and experimental conditions. The analysis confidently assigns a CCS for 49 proteomes, including correct classification for proteomes from synchronized cells. We anticipate that this robust cell cycle protein signature will be crucial for classifying cell states in single-cell proteomes

Biochemical determinants of CDK1 phosphorylation of cell cycle substrates using mass spectrometry-based phospho-proteomics

The cell cycle is a complex series of events that results in one parental cell dividing into two daughter cells. Division only occurs when cells have successfully replicated their DNA, to create progeny each with a copy of their genome. Protein phosphorylation is one mechanism that regulates the cell cycle. Kinases such as the polo-like family, the Auroras, and the Cyclin dependent kinases (CDKs) along with phosphatases, such as PP2A and PP1, form dynamic regulatory circuits that activate and inactivate substrate proteins through reversible phosphorylation of targeted residues. CDK1 is the main regulator of protein phosphorylation in mitosis, carrying out hundreds of these phosphorylations. Monomeric CDK1, however, is inactive and requires either a Cyclin A or Cyclin B partner to gain activity. Progressive expression and degradation of the two Cyclins determine the activity of CDK1 during the cell cycle. Besides, the Cyclin partner was suggested to mediate CDK1 substrate choice. This role, however, has always been a point of debate. Cks1 is another element in CDK1 complexes with even less understood role in substrate phosphorylation. My project aimed to characterise these substrates based on their dependency on CDK1 activity, its Cyclin partner, and the presence of a Cks1 in its complex, in order to understand how the temporal ordering of their phosphorylation is maintained throughout the cell cycle. To achieve that, I developed an in vitro kinase assay through which I phosphorylated fixed and permeabilized cells using recombinant CDK1 in complex with different subunits and assessed their phospho-proteome using mass spectrometry. Results revealed that both CDK1 activity and its Cyclin partner determine the substrate to be targeted for phosphorylation. The presence of Cks1, on the other hand, increased the number of phosphorylated residues on the targeted substrates. This data also unveiled the ability of CDK1 to phosphorylate sites lacking the +1 Proline in its S/TPXK/R consensus motif in the presence of either Cyclin A2 or Cks1 in its complexes. This non-Proline directed phosphorylation uncovered new details of the mechanism by which CDK1 maintains the temporal ordering of substrates phosphorylation. The data here also reveals CDK1 phosphorylation of mitotic sites for which an upstream kinase has not been reported

Small changes in phospho-occupancy at the kinetochore-microtubule interface drive mitotic fidelity

Kinetochore protein phosphorylation promotes the correction of erroneous microtubule attachments to ensure faithful chromosome segregation during cell division. Determining how phosphorylation executes error correction requires an understanding of whether kinetochore substrates are completely (i.e., all-or-none) or only fractionally phosphorylated. Using quantitative mass spectrometry (MS), we measured phospho-occupancy on the conserved kinetochore protein Hec1 (NDC80) that directly binds microtubules. None of the positions measured exceeded ∼50% phospho-occupancy, and the cumulative phospho-occupancy changed by only ∼20% in response to changes in microtubule attachment status. The narrow dynamic range of phospho-occupancy is maintained, in part, by the ongoing phosphatase activity. Further, both Cdk1–Cyclin B1 and Aurora kinases phosphorylate Hec1 to enhance error correction in response to different types of microtubule attachment errors. The low inherent phospho-occupancy promotes microtubule attachment to kinetochores while the high sensitivity of kinetochore–microtubule attachments to small changes in phospho-occupancy drives error correction and ensures high mitotic fidelity