The present invention generally provides, in various embodiments, methods of analyzing samples having a low abundance of proteins, e.g., single cells, utilizing liquid chromatography and tandem mass spectroscopy (LC-MS/MS).

Chemoproteomic methods for detecting and profiling electrophilic post-translational modifications (PTMs) and oxidative PTMs in proteins are described. The methods including contacting a proteomic mixture with a probe having hydrazine and alkyne moieties or oxyamine and alkyne moieties to form a covalent linkage between the hydrazine or oxyamine moiety of the probe and the electrophilic PTM or oxidative PTM of the protein. The resulting alkyne-derivatized proteins are labelled with an azide modified tag via a click chemistry reaction. The labelled proteins can then be detected or profiled using techniques such as, for example, fluorescence imaging or mass spectrometry. Also described are protein conjugates having a covalent linkage formed by reaction of a hydrazine or oxyamine moiety of a probe with an electrophilic or oxidative PTM of a protein.

Bead-based assays for measuring protein biomarkers of proteolytic activity in biological systems are disclosed. In an embodiment, an assay involves incubating a sample containing multiple fragments of a naturally occurring protein with a bead array and subsequently analyzing individual reactive sites of the bead array by mass spectrometry.

The application discloses compositions, methods, systems, and apparatuses for rapid sequence analysis of proteins, including location of post-translational modifications and disulfide bonds. Limited digestion of fully denatured antibody occurs in seconds by flowing sample in 8M urea at constant pressure through a micro column reactor containing immobilized aspergillopepsin I, resulting in a product mixture containing 3-10 kDa peptides, which is then fractionated by capillary column chromatography and analyzed by both electron transfer dissociation (ETD) and collision activated dissociation mass spectrometry. This method provides 95% sequence coverage of a mAb and detects numerous post-translational modifications. For disulfide bond location, native mAb is subjected to longer digestion times. Release of disulfide containing peptides from accessible regions of the folded protein occurs with short digestion times. The identity of peptides connected by a disulfide bond is determined using ETD and ion-ion proton transfer chemistry.

The present invention refers to p53 sequence and post translational modifications (PTMs) and to their use as biomarkers in the diagnosis of neurodegenerative disease and cognitive decline and/or in the prognosis of Alzheimer's disease at different stages and/or of neurodegenerative disease in a biological sample. The invention also provides for a 1) diagnostic method based on a highly accurate mass spectrometry analysis for the diagnosis of neurodegenerative disease, including Mild Cognitive Impairment (MCI), Alzheimer's disease (AD), fronto-temporal dementia (FTD), Lewi's Body (LB), and vascular dementia (VD) in a subject, by evaluating the PTMs to the said p53 linear sequence protein and possible cut of its full sequence specifically in human plasma of patients; and 2) prognosis of AD in CU and MCI patients.

Bead arrays suitable for analysis by mass spectrometry are disclosed. In an embodiment, a bead array includes multiple reactive sites, each of the reactive sites being capable of binding multiple distinct target analytes.

The subject invention pertains to biomarkers for identifying a subject as having high risk of the development PE. The biomarkers presented herein include miRNAs, post-translational modification of histone proteins, amount, expression and/or activity of histone or DNA modifying enzymes and methylation of sites in the genomic DNA. In certain embodiments, increased miR-17, increased acetylation of H4 histone protein, decreased amount, expression and/or activity of HDAC5 mRNA or protein or increased methylation of DNA at the genomic site CYP19A1 in the blood, serum or plasma of a subject compared to that of a control subject is used to predict the development of PE in the subject. The invention also provides kits and reagents to conduct assays to quantify biomarkers described herein. The invention further provides the methods of treating and/or managing PE in a subject identified as having a high risk of the development of PE.

The present invention relates to a method for analysing protein variants of a recombinant protein of interest, such as antibodies or Fc-fusion proteins, in a liquid sample of a mammal. Specifically the method comprises a step of affinity purifying the recombinant protein of interest from the sample together with an internal standard, and analyzing the protein variants using an analytic separating method such as HPLC, capillary electrophoresis or MS. This method is particularly suited to measure pharmacokinetic parameters of a recombinant protein of interest, such as a biopharmaceutical, in a mammal in clinical or pre-clinical studies. It allows for the use of a small sample volume and the possibility to operate with high throughput, such as in a 96-well plate sample preparation. It also provides high sensitivity and allows analysis of protein variants individually.

Antibodies against citrullinated protein antigens (ACPA) have shown their relevance for the diagnosis and possibly pathogenesis in arthritis. Described are means and methods for determining antibodies against homocitrulline-containing proteins or carbamylated proteins/peptides (anti-CarP) for the classification of individuals suffering from, or at risk of suffering from, arthritis.

The present invention features methods for identifying treatments for neurodegenerative diseases, e.g., Parkinson's disease. Described are purification methods for synuclein nitration enzyme and screening methods for identifying agents that inhibit synuclein nitration enzyme activity.