The present description provides methods, assays and reagents useful for sequencing proteins. Sequencing proteins in a broad sense involves observing the plausible identity and order of amino acids, which is useful for sequencing single polypeptide molecules or multiple molecules of a single polypeptide. In one aspect, the methods are useful for sequencing multiple polypeptides. The methods and reagents described herein can be useful for high resolution interrogation of the proteome and enabling ultrasensitive diagnostics critical for early detection of diseases.

A method for increasing peptide fragmentation by labelling the peptide at the C-terminal end with a guanidinium group or other basic functional group and distinguishing isobaric amino acids and amino acid combinations of asparagine and glycine-glycine; glutamine and glycine-alanine; and/or glutamine and alanine-glycine, during polypeptide sequencing. The method involves: obtaining a peptide of interest and/or digesting a polypeptide of interest with a protease, such as pepsin, chymotrypsin or trypsin, or by chemical cleavage to produce shorter peptides; reacting the obtained and/or generated peptides with a coupling reagent to derivatize the free C-terminal carboxylic acid function of the peptides, thus adding a basic functional group rendering C-terminal peptide fragment ions detectable by mass spectrometry; selecting a charge state of 2+ or more, and fragmenting the derivatized peptides in a mass spectrometer under conditions effective to generate at least w ions; and detecting the w ions by mass spectrometry, and identifying derivatized peptides which incorporate the additional mass of the basic functional group.

Aspects of the application provide methods of identifying and sequencing proteins, polypeptides, and amino acids, and compositions useful for the same. In some aspects, the application provides amino acid recognition molecules, such as amino acid binding proteins and fusion polypeptides thereof. In some aspects, the application provides amino acid recognition molecules comprising a shielding element that enhances photostability in polypeptide sequencing reactions.

Methods and devices for preparing target molecules (e.g., target nucleic acids or target proteins) from a biological sample are provided herein. In some embodiments, methods and devices involve sample lysis, sample fragmentation, enrichment of target molecule(s), and/or functionalization of target molecule(s).

The present disclosure provides a rapid, scalable, and high-throughput method of identifying the precise regions in a receptor protein which are involved in binding of a molecule of interest. The method of the instant disclosure is useful where the crystal structure of a protein of interest is not available. Also provided are surface display libraries, and methods of making the same.

The present invention is a method for measuring the amount of at least one molecule in a biological sample, the method comprising a) combining the sample, or a derivative thereof, with one or more aptamers and allowing one or more molecules in the sample to bind to the aptamer(s); b) separating bound from unbound molecules; and c) quantifying the molecule(s) bound to the or each aptamer, wherein quantification of the bound molecule(s) is carried out by sequencing at least part of the or each aptamer. Uses of and products derived from the method are also contemplated.

The present disclosure provides methods of treating subjects having hearing loss, methods of identifying subjects having an increased risk of developing hearing loss, and methods of detecting Kelch Domain Containing 7B (KLHDC7B) variant nucleic acid molecules and variant polypeptides.

The present disclosure provides compositions and methods involving the use of mucin-specific proteases for mucin-specific cleavage, labeling, and/or enrichment of mucin domain glycoproteins. Also provided are methods for the analysis of mucin-domain glycoproteins useful in glycomapping of mucin glycosites and their associated glycoforms. Provided compositions and methods are also useful for selective cleavage, release, and enrichment of mucins from cell and tissue samples, for the study of native mucin biology, and for the detection and analysis of mucins that are aberrantly expressed in various conditions, including cancer.

The present invention provides methods, devices and systems for sequencing and/or analyzing a polymer and/or polymer unit. The polymer may include but not limited to DNA, RNA, a polysaccharide, or a protein. The device includes a nano-pen, which is a bifunctional nanopore/nanoelectrode, and a second electrode. The nano-pen electrode and the second electrode form a tunnel gap. Polymers passing through the nano-pen nanopore will be directed to the tunnel gap between electrodes. The electrodes are functionalized with a recognition reagent, and the reagent can transiently bind each polymer unit during its passage. When the transient bond forms, distinctive current signals are detected and recorded. The signals are utilized to analyze and identify the polymer and/or polymer unit.

The present disclosure generally relates in certain embodiments to the creation of ionized molecules, e.g., for detection in a mass spectrometer, or for other uses such as lithography, sputtering machines, propulsion etc. Some embodiments include an ion source comprising a capillary tip that may allow for direct ion evaporation of samples with an applied electric field. In some cases, the tip may have an opening with a cross-section less than 100 nm. In addition, certain aspects are directed to using a capillary tip that allow for detection of samples (e.g. amino acids), and in some cases allows for sequencing. For instance, some embodiments are directed to allowing single ions and ionic clusters to be evaporated at a high rate directly from aqueous samples in a mass spectrometer. Other aspects are directed to methods for making or using such ionized molecules, methods for making or using devices to create such ionized molecules, or the like.