High Resolution Discovery Proteomics Reveals Candidate Disease Progression Markers of Alzheimer's Disease in Human Cerebrospinal Fluid.

Disease modifying treatments for Alzheimer's disease (AD) constitute a major goal in medicine. Current trends suggest that biomarkers reflective of AD neuropathology and modifiable by treatment would provide supportive evidence for disease modification. Nevertheless, a lack of quantitative tools to assess disease modifying treatment effects remains a major hurdle.

Quantitative analysis of apolipoproteins in human HDL by top-down differential mass spectrometry.

The field of quantitative, label-free proteomics has evolved significantly over time, with most experiments performed "bottom-up" using proteolyzed protein mixtures. In these experiments, statistically significant peptide abundance differences between two or more experimental conditions are determined, and their corresponding proteins later identified. Recently, the rationale for extending this experimental design to mixtures of intact proteins has become clear, as analysis at the protein level allows for the independent detection of each protein form present, including those modified posttranslationally.

A platform for characterizing therapeutic monoclonal antibody breakdown products by 2D chromatography and top-down mass spectrometry.

With the growing commercialization of therapeutic monoclonal antibodies developed for the treatment of various diseases comes the need for increased analytical scrutiny of the impurity components contained within such drug products. Traditionally, relatively low performance and throughput analytical techniques were employed for elucidating the product-related breakdown components derived from the original molecule, including N-terminal Edman sequencing and matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry. Although N-terminal sequencing provides a definitive starting point of an unknown breakdown product, the resolution and mass accuracy of MALDI-TOF instruments are often insufficient for unambiguous sequence characterization.

An algorithm for identifying multiply modified endogenous proteins using both full-scan and high-resolution tandem mass spectrometric data.

Mass spectrometry based proteomic experiments have advanced considerably over the past decade with high-resolution and mass accuracy tandem mass spectrometry (MS/MS) capabilities now allowing routine interrogation of large peptides and proteins. Often a major bottleneck to 'top-down' proteomics, however, is the ability to identify and characterize the complex peptides or proteins based on the acquired high-resolution MS/MS spectra. For biological samples containing proteins with multiple unpredicted processing events, unsupervised identifications can be particularly challenging.

Quantitative analysis of intact apolipoproteins in human HDL by top-down differential mass spectrometry.

Top-down mass spectrometry holds tremendous potential for the characterization and quantification of intact proteins, including individual protein isoforms and specific posttranslationally modified forms. This technique does not require antibody reagents and thus offers a rapid path for assay development with increased specificity based on the amino acid sequence. Top-down MS is efficient whereby intact protein mass measurement, purification by mass separation, dissociation, and measurement of product ions with ppm mass accuracy occurs on the seconds to minutes time scale.

Quantitative analysis of complex peptide mixtures using FTMS and differential mass spectrometry.

Label-free LC-MS profiling is a powerful quantitative proteomic method to study relative peptide abundances between two or more biological samples. Here we demonstrate the use of a previously described comparative LC-MS method, differential mass spectrometry (dMS), to analyze high-resolution Fourier transform mass spectrometry (FTMS) data for detection and quantification of known peptide differences between two sets of complex mixtures. Six standard peptides were spiked into a processed plasma background at fixed ratios from 1.

Chemoenzymatic approaches for streamlined detection of active site modifications on thiotemplate assembly lines using mass spectrometry.

For the direct interrogation of peptides harboring covalently modified serines in nonribosomal peptide synthetases, streamlined methodologies described here employ proteolysis and reporter-coenzyme A analogues of four types. The chromophoric and fluorescent coenzyme A analogues pyrene-maleimidyl-S-CoA and BODIPY-FL-N-(2-aminoethyl)maleimidyl-S-CoA were enzymatically loaded onto the active site serines harbored in the ArCP, PCP1, and PCP2 thiolation domains of PchE and PchF, the nonribosomal peptide synthetases responsible for the biosynthesis of the siderophore pyochelin. During the chromatographic separation of cyanogen bromide digests, observation of the absorbance (at 338 and 504 nm) or fluorescence (after irradiation at 365 nm) enabled the selective detection of peptides containing each active site serine.

Parallel interrogation of covalent intermediates in the biosynthesis of gramicidin S using high-resolution mass spectrometry.

For determination of multiple covalent intermediates bound to the ultra-large enzymes responsible for biosynthesis via nonribosomal peptide synthesis, mass spectrometry (MS) is a promising method to provide new mechanistic insight. Application of a quadrupole-Fourier-transform instrument (Q-FTMS) for direct analysis of aminoacyl intermediates is demonstrated for the first two modules (127 and 120 kDa) involved in the nonribosomal synthesis of gramicidin S. Cyanogen bromide digestions of recombinant proteins afforded detection of two active site peptides (both ~13 kDa) that provided direct evidence for modules copurifying with their preferred amino acid substrates.

Mass spectrometric interrogation of thioester-bound intermediates in the initial stages of epothilone biosynthesis.

Direct detection of thioester intermediate mixtures bound to EpoC, a 195 kDa polyketide synthase, has been achieved using limited proteolysis and Fourier-transform mass spectrometry (FTMS). Incubation with various N-acetylcysteamine thioester (S-NAC) substrate mimics produced mass shifts on the EpoC ACP domain consistent with their condensation with an enzyme-bound carbanion produced by the decarboxylation of methylmalonyl-S-EpoC. Reconstitution of EpoA-ACP, EpoB, and EpoC gave a +165.

Site-specific observation of acyl intermediate processing in thiotemplate biosynthesis by fourier transform mass spectrometry: the polyketide module of yersiniabactin synthetase.

Complex arrays of thioester bound intermediates are present on 100-700 kDa enzymes during the biogenesis of diverse types of pharmacophores and natural product drugs. These multidomain enzymes, known as nonribosomal peptide synthetases and polyketide synthases (NRPSs and PKSs, respectively), synthesize from simple, physiologically available substrates bioactive compounds that can be further tailored by a host of modifying domains (e.g.