Single-molecule library preparation of a complex cell lysate

Protein Identification by Short-epitope Mapping (PrISM) is a novel, single-molecule based proteome analysis approach that is designed to quantify nearly the entire human proteome in a single experiment. A key attribute of this approach includes the ability to provide comprehensive coverage of sample constituents and a wide dynamic range suitable to quantify high- and low-abundant proteins in the proteome. The first step in PrISM involves the generation of a single-molecule library from the sample, which must be simple-to-execute, efficient, reproducible and comprehensive to consistently provide a representative portion of the sample for subsequent deposition and detection on the billions of landing pads in the flowcell used for analysis.

Using both a model-protein mixture and a U2-OS cell line, we prepared a library of each mixture by denaturing the sample using SDS and functionalizing the lysine residues and N-terminal primary amines using an STP methyltetrazine group (mTz), which puts a click reagent onto each of the proteins. To complete the single pot reaction, we reduced and alkylated the disulfide bonds. Cleanup steps removed the reactants, resulting in protein molecules labeled with the click group mTz. These labeled proteins were then conjugated to a proprietary nanoparticle scaffold for deposition onto our hyper dense flowcell.

We first investigated the efficiency of labeling model-protein mixture and lysate samples with STP-mTz by applying bottom-up LCMS to the modified samples. As we expect many lysine residues to be modified, samples were digested with both trypsin and arginase-C to maximize sequence coverage. We obtained a broad coverage of the U2-OS proteome, providing a direct read out of the lysine residue and N-terminal primary amine modifications on the lysate. The results indicate that, on average, more than one lysine residue per protein is modified by mTz allowing for the efficient covalent reaction with the complimentary click group TCO on the nanoparticle scaffolds. We did not see any obvious classes of proteins that were unable to be modified. We next investigated the efficiency of conjugation between mTz modified proteins and nanoparticle scaffolds through LCMS analysis of the proteins that had been successfully conjugated to the nanoparticles. Comparing our U2-OS LCMS analysis with previously published U2-OS proteome analysis under similar LCMS conditions indicates that our simple, straightforward library preparation method enables the U2-OS proteome to be deposited as single proteins at high density on the flowcell for subsequent analysis by PrISM.