A method for analyzing macromolecules, including peptides, polypeptides, and proteins, employing nucleic acid encoding is disclosed.

Kits and methods of using the kits for analyzing macromolecules, including peptides, polypeptides, and proteins, employing nucleic acid encoding are disclosed. The sample analysis kits employ nucleic acid encoding and/or nucleic acid recording of a molecular interaction and/or reaction, such as recognition events (e.g., between an antigen and an antibody, between a modified terminal amino acid residue, or between a small molecule or peptide therapeutic and a target, etc.). Additional barcoding reagents, such as those for cycle-specific barcoding (e.g., clocking), compartment barcoding, combinatorial barcoding, spatial barcoding, or any combination thereof, may be included in the kits. The sample may comprise macromolecules, including peptides, polypeptides, and proteins, and the recording may generate molecular interaction and/or reaction information, and/or polypeptide sequence information. The kits may be used in high-throughput, multiplexed, and/or automated analysis, and are suitable for analysis of a proteome or subset thereof.

The presently disclosed subject matter provides methods using mass spectrometry for direct profiling of N-linked glycans from a biological sample. In addition, the embodiments of the present invention also disclose novel methods, known as targeted analyte detection (TAD), for improving the detection limit of MALDI-MS. These methods take advantage of the carrier effect of the added standard analytes, which occurs due to the generic sigmoidal shape of the calibration curve. The functionality of TAD depends on the relative enhancement of sensitivity over the increase of the standard deviation at the analysis of target analytes with spiking in exogenous concentration. At certain ranges of exogenous concentration, the increment in the sensitivity overcomes the standard deviation, resulting in an improved LOD. Theoretically, exogenous concentrations approximately at 1 LODorig would generate the optimum LOD improvement. TAD is a cost-effective LOD improvement method, which is not limited to a certain group of analytes, or detection methods or instruments. It can be applied to enhance the detection of any analyte with different detection methods, provided that the analyte of interest can be extracted or is available in synthetic form.

The state of protein phosphorylation and glycosylation can be key determinants of cellular physiology such as early stage cancer, but the development of phosphoproteins and/or glycoproteins in biofluids for disease diagnosis remains elusive. Here we demonstrate, for the first time, a strategy to isolate and identify phosphoproteins/glycoproteins in extracellular vehicles (EVs) from human plasma as potential markers to differentiate disease from healthy states. We identified close to 10,000 unique phosphopeptides in EVs by isolating from small volume of plasma samples. Using label-free quantitative phosphoproteomics, we identified 144 phosphoproteins in plasma EVs that are significantly higher in patients diagnosed with breast cancer than in healthy controls. Several novel biomarkers were validated in individual patients using Paralleled Reaction Monitoring for targeted quantitation. Similarly a group of glycoproteins in plasma EVs are identified. The study demonstrated that the development of phosphoproteins and/or glycoproteins in plasma EV as disease biomarkers is highly feasible and may transform cancer screening and monitoring.

The present invention provides compositions comprising chimeric polypeptides that bind to free ubiquitin proteins or free ubiquitin-like proteins with high affinity, as well as chimeric polypeptides that bind to both free and conjugated ubiquitin proteins or free and conjugated ubiquitin-like proteins, and methods of using the chimeric polypeptides to determine the amount of free or total ubiquitin or free or total ubiquitin-like proteins in various types of samples.

Methods of analyzing glycosylated biomolecules include the steps of producing a deglycosylation mixture of biomolecules deglycosylated by natural or synthetic enzymatic or chemical techniques; providing a reagent solution having a labeling reagent in a polar aprotic, non-nucleophilic organic solvent; and mixing the deglycosylation mixture with the reagent solution in an excess of labeling reagent to produce derivatized glycosylamines. The method steps can be carried out purposefully without depletion of protein matter. A quenching solution can be added to the reaction mixture so that the pH of the reaction mixture is shifted to above 10. The yield of derivatized glycosylamines can be in an amount of about 80 to about 100 mole percent of the reaction mixture with minimal overlabeling, less than 0.2 mole percent. The derivizated glycosylamines can be separated from the reaction mixture and detected by chromatographic detection, fluorescence detection, mass spectrometry (MS), or Ultra Violet (UV) detection and/or a combination thereof.

The present invention relates to the methods of developing recombinant proteins with a fingerprint like similarity to the reference product or the originator. The method is particularly useful in the development of biosimilar products. This method can also be used to establish comparability during the manufacturing process change for the originator products. Hie methods described herein are used to obtain a recipe for the production of a biosimilar product or a recombinant protein using a process that may be different from the original but that yields a recombinant protein that has fingerprint level of similarity to the reference product. The methods described herein can also used to obtain a fingerprinting analysis package for a biosimilar that can be submitted to regulatory agency for abbreviated biosimilar approval. While currently available analytical methods can identify and quantitate specific modifications on a recombinant, protein, no methods currently exist to measure and determine the concentration of product variants in a complex: mixture. The analytical methods described herein provide for identification and quantitation of the modifications of the recombinant proteins and of product variants in a complex mixture by utilizing various in silico computational approaches to transform analytical data and derive product variant distribution.

The present invention concerns a method for identifying and using a biomarker, or creating a proteome profile, indicative of a reduced response to a drug in a patient involving a thermal shift assay on a sample. The method comprises the steps of a) heating a sample from a patient b) separating soluble from insoluble protein, c) analysing either or both the soluble and insoluble protein fractions of step b) to determine the melting temperature.

Fructosyl amino acid oxidase is provided, which has an amino acid sequence as shown in SEQID. No. 1 or fructosyl amino acid oxidase having a homology of more than 80% with this amino acid sequence, on a corresponding site of an amino acid selected from following (a) to (f), having one or more amino acid residues conducting a substitution, obtained fructosyl amino acid oxidase having a higher thermostability: (a) 59-site glutamic acid, (b) 98-site glutamic acid, (c) 225-site glycine, (d) 277-site lysine, (e) 285-site glutamic acid, and (f) 355-site aspartic acid. The method for preparing the above oxidase and the test kit containing the enzyme for determining glycated albumin are also provided.

The present disclosure provides a system comprising a communication interface and computer for assigning a label to the biomolecule fingerprint, wherein the label corresponds to a biological state. The present disclosure also provides a sensor arrays for detecting biomolecules and methods of use. In some embodiments, the sensor arrays are capable of determining a disease state in a subject.