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Proton-transfer reactions (PTRs) have emerged as a powerful tool for the study of intact proteins. When coupled with m/z-selective kinetic excitation, such as parallel ion parking (PIP), one can exert exquisite control over rates of reaction with a high degree of specificity. This allows one to "concentrate", in the gas phase, nearly all the signals from an intact protein charge state envelope into a single charge state, improving the signal-to-noise ratio (S/N) by 10× or more. While this approach has been previously reported, here we show that implementing these technologies on a 21 T FT-ICR MS provides a tremendous advantage for intact protein analysis. Advanced strategies for performing PTR with PIP were developed to complement this unique instrument, including subjecting all analyte ions entering the mass spectrometer to PTR and PIP. This experiment, which we call "PTR-MS1-PIP", generates a pseudo-MS1 spectrum derived from ions that are exposed to the PTR reagent and PIP waveforms but have not undergone any prior true mass filtering or ion isolation. The result is an extremely rapid and significant improvement in the spectral S/N of intact proteins. This permits the observation of many more proteoforms and reduces ion injection periods for subsequent tandem mass spectrometry characterization. Additionally, the product ion parking waveform has been optimized to enhance the PTR rate without compromise to the parking efficiency. We demonstrate that this process, called "rapid park", can improve reaction rates by 5-10× and explore critical factors discovered to influence this process. Finally, we demonstrate how coupling PTR-MS1 and rapid park provides a 10-fold reduction in ion injection time, improving the rate of tandem MS sequencing.

MetaMorpheus is a free, open-source software program for the identification of peptides and proteoforms from data-dependent acquisition tandem MS experiments. There is inherent uncertainty in these assignments for several reasons, including the limited overlap between experimental and theoretical peaks, the m/z uncertainty, and noise peaks or peaks from coisolated peptides that produce false matches. False discovery rates provide only a set-wise approximation for incorrect spectrum matches. Here we implemented a binary decision tree calculation within MetaMorpheus to compute a posterior error probability, which provides a measure of uncertainty for each peptide-spectrum match. We demonstrate its utility for increasing identifications and resolving ambiguities in bottom-up, top-down, proteogenomic, and nonspecific digestion searches.

Identification of proteoforms, the different forms of a protein, is important to understand biological processes. A proteoform family is the set of different proteoforms from the same gene. We previously developed the software program Proteoform Suite, which constructs proteoform families and identifies proteoforms by intact-mass analysis. Here, we have applied this approach to top-down proteomic data acquired at the National High Magnetic Field Laboratory 21 tesla Fourier transform ion cyclotron resonance mass spectrometer (data available on the MassIVE platform with identifier MSV000085978). We explored the ability to construct proteoform families and identify proteoforms from the high mass accuracy data that this instrument provides for a complex cell lysate sample from the MCF-7 human breast cancer cell line. There were 2830 observed experimental proteforms, of which 932 were identified, 44 were ambiguous, and 1854 were unidentified. Of the 932 unique identified proteoforms, 766 were identified by top-down MS2 analysis at 1% false discovery rate (FDR) using TDPortal, and 166 were additional intact-mass identifications (∼4.7% calculated global FDR) made using Proteoform Suite. We recently published a proteoform level schema to represent ambiguity in proteoform identifications. We implemented this proteoform level classification in Proteoform Suite for intact-mass identifications, which enables users to determine the ambiguity levels and sources of ambiguity for each intact-mass proteoform identification.

The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product's primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information on the common TD/MD MS methods and practices in the field. A panel of three mAbs was selected and centrally provided to 20 laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). Various MS instrument platforms and ion dissociation techniques were employed. The present study confirms that TD/MD MS tools are available in laboratories worldwide and provide complementary information to the BU approach that can be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. A primary limitation revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS.

The Consortium for Top-Down Proteomics (CTDP) proposes a standardized notation, ProForma, for writing the sequence of fully characterized proteoforms. ProForma provides a means to communicate any proteoform by writing the amino acid sequence using standard one-letter notation and specifying modifications or unidentified mass shifts within brackets following certain amino acids. The notation is unambiguous, human-readable, and can easily be parsed and written by bioinformatic tools. This system uses seven rules and supports a wide range of possible use cases, ensuring compatibility and reproducibility of proteoform annotations. Standardizing proteoform sequences will simplify storage, comparison, and reanalysis of proteomic studies, and the Consortium welcomes input and contributions from the research community on the continued design and maintenance of this standard.

A hybridization-based FRET format for the scoring of SNPs in surface invasive cleavage reactions is described. In early versions of the surface invasive cleavage reaction, dual-labeled oligonucleotides, containing both a quencher moiety and a fluorophore, were attached to the substrate. The invasive cleavage reaction cleaved the DNA strand between the two, resulting in an increase in fluorescence signal due to the separation of the quencher from the fluorophore. A limitation of this assay format was the relatively low quenching efficiency of 84% obtained, as well as the complexity of synthesis for these dual-labeled probes. In the assay format presented here, singly labeled oligonucleotides are employed, with the quencher and fluorophore placed on separate complementary oligonucleofides. The surface-bound probe is terminated at the 5' end with the quencher and the complement is terminated at its 3' end with a fluorophore, such that upon hybridization the two are positioned directly across from one another in the duplex. Quenching efficiency in this 'molecular beacon' format is increased to 88%, much closer to the 91% level that has been reported for molecular beacon assays. A second benefit of the approach described here is that the portion of probe oligonucleotide that is removed by the enzyme is shorter, thus increasing the rate of probe cleavage. The improved quenching efficiency and increased probe cleavage rate result in a lower detection limit for the assay. A theoretical model of the FRET process occurring on the surfaces was used to relate the observed surface fluorescence intensity to the progress of the invasive cleavage reaction.

Cell surface carbohydrates play important roles in a wide variety of biological processes including cell adhesion, fertilization, differentiation, development, and tumor cell metastasis. Lectins are proteins of nonimmune origin which recognize and bind to specific carbohydrate structural epitopes. We have recently described the development and use of lectin arrays as tools for the elucidation of the carbohydrate structures expressed on cell surfaces. In the present work this technology is employed for the characterization of differences in carbohydrate expression patterns on normal and tumorigenic human breast cell lines, as well as on sublines differing in their tendency to 'home' to different tissues during metastasis. Significant differences were observed, including changes that correlate with metastatic potential as well as with tissuespecific homing of metastatic cells.

Labeling reagents that differ only in their isotopic composition offer a powerful approach to achieve relative quantification between samples by ESI-MS. Heavy and light isotopic forms of cholamine, which contain a positively charged quaternary ammonium group, were synthesized and tested as new labeling reagents for the relative quantification of carboxylic acid-containing metabolites, specifically fatty acids. The positive charge on cholamine ensures that the labeled product is also positively charged under all LC-MS conditions, regardless of mobile-phase pH. This leads to high ionization efficiency and correspondingly high detection sensitivity, demonstrated here for the analysis of fatty acids in positive ion mode ESI-MS after reversed-phase separation under acidic conditions. Good accuracy and precision were obtained by mixing heavy- and light-labeled hydrolyzed egg lipid extracts in different known ratios. The relative quantification results for 10 observed fatty acids had an average absolute error of 4.6% and an average coefficient of variation (CV) of 2.6%. The labeling strategy yielded a median CV of 6% when employed for fatty acid analysis of eggs from chickens fed various dietary supplements.

A powerful approach to relative quantification by mass spectrometry is to employ labeling reagents that target specific functional groups in molecules of interest. A quantitative comparison of two or more samples may be readily accomplished by using a chemically identical but isotopically distinct labeling reagent for each sample. The samples may then be combined, subjected to purification steps, and mass analyzed. Comparison of the signal intensities obtained from the isotopically labeled variants of the target analyte(s) provides quantitative information on their relative concentrations in the sample. In this report, we describe the synthesis and use of heavy and light isotopic forms of methyl acetimidate for the relative quantification of amine-containing species. The principal advantages of methyl acetimidate as a labeling reagent are that the reaction product is positively charged and hydrophobicity is increased, both of which enhance electrospray ionization efficiency and increase detection sensitivity. The quantitative nature of the analysis was demonstrated in model metabolomics experiments in which heavy and light labeled Arabidopsis extracts were combined in different ratios. Finally, the labeling strategy was employed to determine differences in the amounts of amine-containing metabolites for Arabidopsis seeds germinated under two different conditions.

Single-nucleotide polymorphisms (SNPs) are the most frequent type of human genetic variation. Recent work has shown that it is possible to directly analyze SNPs in unamplified human genomic DNA samples using the surface-invasive cleavage reaction followed by rolling circle amplification (RCA) labeling of the cleavage products. The individual RCA amplicon molecules were counted on the surface using fluorescence microscopy. Two principal limitations of such single-molecule counting are the variability in the amplicon size, which results in a large variation in fluorescence signal intensity from the dye-labeled DNA molecules, and a high level of background fluorescence. It is shown here that an excellent alternative to RCA labeling is tagging with gold nanoparticles followed by imaging with a scanning electron microscope. Gold nanoparficles have a uniform diameter (15 [+ or -] 0.5 nm) and provide excellent contrast against the background of the silicon substrate employed. Individual gold nanoparticles are readily counted using publicly available software. The results demonstrate that the labeling efficiency is improved by as much as ~15-fold, and the signal-to-noise ratio is improved by ~4-fold. Detection of individual cleaved DNA molecules following surface-invasive cleavage was linear and quantitative over 3 orders of magnitude in amount of target DNA ([10.sup.-18]-[10.sup.-15] mol).

Single-nucleotide polymorphisms (SNPs) are the most frequent type of human genetic variation. Recent work has shown that it is possible to directly analyze SNPs in unamplified human genomic DNA samples using the surface-invasive cleavage reaction followed by rolling circle amplification (RCA) of the cleavage products. The ability of RCA to produce single-stranded DNA tens of thousands of nucleotides in length from a single cleaved DNA molecule on the surface suggested the possibility of detecting individual cleavage events on the surface. The feasibility of this approach to SNP scoring is shown here. Individual cleavage events on the surface are detected using fluorescence microscopy to visualize the singlestranded DNA product of the RCA reaction labeled with the fluorescent dye SYBR Green I. The surface density of fluorescent features observed is dependent upon the concentration of target DNA. Future reductions of the sample volume and optimization of the reaction conditions offer the potential of being able to perform such analyses on as little as a single copy of genomic DNA target.

Most patients at risk for developing type 2 diabetes are hyperinsulinemic. Hyperinsulinemia may be a response to insulin resistance, but another possible abnormality is insulin hypersecretion. BTBR mice are insulin resistant and hyperinsufinemic. When the [leptin.sup.ob] mutation is introgressed into BTBR mice, they develop severe diabetes. We compared the responsiveness of lean B6 and BTBR mouse islets to various insulin secretagogues. The transamination product of leucine, [alpha]-ketoisocaproate (KIC), elicited a dramatic insulin secretory response in BTBR islets. The KIC response was blocked by methyl-leucine or aminooxyacetate, inhibitors of branched-chain amino transferase. When dimethylglutamate was combined with KIC, the fractional insulin secretion was identical in islets from both mouse strains, predicting that the amine donor is rate-limiting for KIC-induced insulin secretion. Consistent with this prediction, glutamate levels were higher in BTBR than in B6 islets. The transamination product of glutamate, [alpha]-ketoglutarate, elicited insulin secretion equally from B6 and BTBR islets. Thus formation of [alpha]-ketoglutarate is a requisite step in the response of mouse islets to KIC. [alpha]-Ketoglutarate can be oxidized to succinate. However, succinate does not stimulate insulin secretion in mouse islets. Our data suggest that [alpha]-ketoglutarate may directly stimulate insulin secretion and that increased formation of [alpha]-ketoglutarate leads to hyperinsulinemia. [beta]-cells; hyperinsulinemia; glutamate dehydrogenase; branched-chain aminotransferase

Large-scale investigations of sequence variation within the human species will provide information about the basis of heritable variation in disease susceptibility and human migration. The surface invader assay (an adaptation of the invasive cleavage reaction to an array format) is capable of exquisitely sensitive and specific detection of genetic variation. It is shown here that this genotyping technology can be multiplexed in a DNA array format, permitting the parallel analysis of a panel of single nucleotide polymorphisms (SNPs) directly from an unamplified genomic DNA target. In addition, a 'universal' mode of detection was developed that makes use of a mixture of degenerate templates for DNA ligation to the surface-bound cleaved oligonucleotides and thereby makes this strategy amenable to any desired SNP site or combination of SNP sites, without regard to their particular DNA sequences. This approach was demonstrated on a proof-of-principle scale using small DNA arrays to genotype 6 SNP markers in the PTPN1 gene and 10 mutations in the cystic fibrosis transmembrane conductance regulator gene. This ability to analyze many different genetic variations in parallel, directly from unamplified human genomic DNA samples, lays the groundwork for the development of high-density arrays able to analyze hundreds of thousands or even millions of SNPs.

In electrophoresis, the migration velocity is used for sizing DNA and proteins or for distinguishing molecules based on charge and hydrodynamic radius. Many protein and DNA assays relevant to disease diagnosis are based on such separations. However, standard protocols are not only slow (minutes to hours) but also insensitive (many molecules in a detectable band). We successfully demonstrated a high-throughput imaging approach that allows determination of the individual electrophoretic mobilities of many molecules at a time. Each measurement only requires a few milliseconds to complete. This opens up the possibility of screening single copies of DNA or proteins within single biological cells for disease markers without performing polymerase chain reaction or other biological amplification. The purpose is not to separate the DNA molecules but to identify each one on the basis of the measured electrophoretic mobility. We developed three different procedures to measure the individual molecular mobilities. The results correlate well with capillary electrophoresis (CE) experiments for the same samples (2-49 kb dsDNA) under identical separation conditions. The implication is that any electrophoresis protocols from slab gels to CE should be adaptable to single-molecule screening for disease diagnosis.

Lipophilic anionic and cationic additives are investigated as components for use in liquid polymer film-based optodes. These ionic additives are known to strongly influence the selectivity behavior of anion-selective electrodes. They also make it possible to construct ion coextraction optodes that can measure anion activities. Theoretical, thermodynamic anion-optode equilibria formalisms were derived to help understand the influence these additives have on the overall optode response and on the selectivity behavior. Neutral and charged anion carrier film configurations are described and tested for two anion ionophores with known modes of action, ruthenium(II) octaethylporphyrin and a vitamin [B.sub.12] derivative (cyanoaquacobyrinic acid heptakis(2-phenylethyl ester)). These film configurations were further tested using a less well-understood ionophore, indium(III) octaethylporphyrin. Notable is the indium(III) octaethylporphyrin optode, which has a dynamic range appropriate for physiological measurements of chloride at neutral pH values. Also of interest is the optode with the vitamin derivative ionophore, which has a dynamic range appropriate for some physiological measurements of nitrite at neutral pH values.