Methods of analyzing a drug polypeptide conjugate using tandem mass spectrometry involving dual tandem mass tags (TMTs) are described. Provided herein is an integrated method using TMTs to obtain three analytical measurements, termed “triple play”, which enables identification of the drug occupancy, normalization between multiple samples and triggering of additional MS/MS to identify and localize conjugation site(s) of the payload. Also described is a method of multiplexing conjugation reactions in a single run with TMT labeling for enhanced throughput capability, while maintaining the same sensitivity with current mass spectrometry instrumentation.

Methods of analyzing a drug polypeptide conjugate using tandem mass spectrometry involving dual tandem mass tags (TMTs) are described. Provided herein is an integrated method using TMTs to obtain three analytical measurements, termed “triple play”, which enables identification of the drug occupancy, normalization between multiple samples and triggering of additional MS/MS to identify and localize conjugation site(s) of the payload. Also described is a method of multiplexing conjugation reactions in a single run with TMT labeling for enhanced throughput capability, while maintaining the same sensitivity with current mass spectrometry instrumentation.

In some embodiments, a mass spectrometry tag may comprise a linker region, a mass balance region, and a reporter region. The mass spectrometry tag may be configured to fragment in a mass spectrometer via an energy dependent process to produce multiple reporter molecules. For example, the reporter region of the tag may be configured to produce at least two reporter molecules via fragmentation. In some embodiments, one or more regions of the tag may comprise at least one heavy isotope. In some such embodiments, the ability to fragment into multiple reporter molecules as well as the placement and/or number of heavy isotope(s) allows the mass spectrometry tag to be distinguished from other similar mass spectrometry tags. In some such embodiments, the ability to distinguish between tags having the same or substantially similar total mass to charge ratio and reporter region mass may allow the system to have a greater multiplexing capacity than conventional systems.

In some embodiments, a mass spectrometry tag may comprise a linker region, a mass balance region, and a reporter region. The mass spectrometry tag may be configured to fragment in a mass spectrometer via an energy dependent process to produce multiple reporter molecules. For example, the reporter region of the tag may be configured to produce at least two reporter molecules via fragmentation. In some embodiments, one or more regions of the tag may comprise at least one heavy isotope. In some such embodiments, the ability to fragment into multiple reporter molecules as well as the placement and/or number of heavy isotope(s) allows the mass spectrometry tag to be distinguished from other similar mass spectrometry tags. In some such embodiments, the ability to distinguish between tags having the same or substantially similar total mass to charge ratio and reporter region mass may allow the system to have a greater multiplexing capacity than conventional systems.

Described herein are methods for selectively cleaving the C-terminal amino acid of a peptide or protein. The methods described herein may be applicable for, for example, single-molecule peptide or protein sequencing.

Described herein are methods for selectively cleaving the C-terminal amino acid of a peptide or protein. The methods described herein may be applicable for, for example, single-molecule peptide or protein sequencing.

Aspects of the present disclosure include methods of producing modified polypeptides and modified polypeptide-ribosome or polypeptide-mRNA complexes, and methods of screening polynucleotide and polypeptide libraries. The present disclosure also provides polypeptide libraries useful in screening for single molecule phenotypes. Also provided are kits useful for producing polypeptides capable of being modified using methods disclosed herein.

Aspects of the present disclosure include methods of producing modified polypeptides and modified polypeptide-ribosome or polypeptide-mRNA complexes, and methods of screening polynucleotide and polypeptide libraries. The present disclosure also provides polypeptide libraries useful in screening for single molecule phenotypes. Also provided are kits useful for producing polypeptides capable of being modified using methods disclosed herein.

Covalently cross-linked pilus polymers displayed on the cell surface of Gram-positive bacteria are assembled by class C sortase enzymes. These pilus-specific transpeptidases located on the bacterial membrane catalyze a two-step protein ligation reaction—first, cleaving the LPXTG motif of one pilin protomer to form an acyl-enzyme intermediate, and second, joining the terminal threonine to the nucleophilic lysine residue residing within the pilin motif of another pilin protomer. Informed by the high-resolution crystal structures of corynebacterial pilus-specific sortase (SrtA) and by developing structural variants of the sortase enzyme whose catalytic pocket has been unmasked by activating mutations, we have developed new reagents capable of forming isopeptide bonds in vitro. The reagents disclosed herein can catalyze ligation of isolated SpaA domains in vitro provide a facile and versatile new platform for protein engineering and bio-conjugation that has major implications for biotechnology.

Covalently cross-linked pilus polymers displayed on the cell surface of Gram-positive bacteria are assembled by class C sortase enzymes. These pilus-specific transpeptidases located on the bacterial membrane catalyze a two-step protein ligation reaction—first, cleaving the LPXTG motif of one pilin protomer to form an acyl-enzyme intermediate, and second, joining the terminal threonine to the nucleophilic lysine residue residing within the pilin motif of another pilin protomer. Informed by the high-resolution crystal structures of corynebacterial pilus-specific sortase (SrtA) and by developing structural variants of the sortase enzyme whose catalytic pocket has been unmasked by activating mutations, we have developed new reagents capable of forming isopeptide bonds in vitro. The reagents disclosed herein can catalyze ligation of isolated SpaA domains in vitro provide a facile and versatile new platform for protein engineering and bio-conjugation that has major implications for biotechnology.