Accurate Peptide Fragment Mass Analysis: Multiplexed Peptide Identification and Quantification

Fourier transform-all reaction monitoring (FT-ARM) is a novel approach for the identification and quantification of peptides that relies upon the selectivity of high mass accuracy data and the specificity of peptide fragmentation patterns. An FT-ARM experiment involves continuous, data-independent, high mass accuracy MS/MS acquisition spanning a defined <i>m</i>/<i>z</i> range. Custom software was developed to search peptides against the multiplexed fragmentation spectra by comparing theoretical or empirical fragment ions against every fragmentation spectrum across the entire acquisition. A dot product score is calculated against each spectrum to generate a score chromatogram used for both identification and quantification. Chromatographic elution profile characteristics are not used to cluster precursor peptide signals to their respective fragment ions. FT-ARM identifications are demonstrated to be complementary to conventional data-dependent shotgun analysis, especially in cases where the data-dependent method fails because of fragmenting multiple overlapping precursors. The sensitivity, robustness, and specificity of FT-ARM quantification are shown to be analogous to selected reaction monitoring-based peptide quantification with the added benefit of minimal assay development. Thus, FT-ARM is demonstrated to be a novel and complementary data acquisition, identification, and quantification method for the large scale analysis of peptides

Mango: A General Tool for Collision Induced Dissociation-Cleavable Cross-Linked Peptide Identification

Chemical cross-linking combined with mass spectrometry provides a method to study protein structures and interactions. The introduction of cleavable bonds in a cross-linker provides an avenue to decouple released peptide masses from their precursor species, greatly simplifying the downstream search, allowing for whole proteome investigations to be performed. Typically, these experiments have been challenging to carry out, often utilizing nonstandard methods to fully identify cross-linked peptides. Mango is an open source software tool that extracts precursor masses from chimeric spectra generated using cleavable cross-linkers, greatly simplifying the downstream search. As it is designed to work with chimeric spectra, Mango can be used on traditional high-resolution tandem mass spectrometry (MS/MS) capable mass spectrometers without the need for additional modifications. When paired with a traditional proteomics search engine, Mango can be used to identify several thousand cross-linked peptide pairs searching against the entire <i>Escherichia coli</i> proteome. Mango provides an avenue to perform whole proteome cross-linking experiments without specialized instrumentation or access to nonstandard methods

Mango: A General Tool for Collision Induced Dissociation-Cleavable Cross-Linked Peptide Identification

Chemical cross-linking combined with mass spectrometry provides a method to study protein structures and interactions. The introduction of cleavable bonds in a cross-linker provides an avenue to decouple released peptide masses from their precursor species, greatly simplifying the downstream search, allowing for whole proteome investigations to be performed. Typically, these experiments have been challenging to carry out, often utilizing nonstandard methods to fully identify cross-linked peptides. Mango is an open source software tool that extracts precursor masses from chimeric spectra generated using cleavable cross-linkers, greatly simplifying the downstream search. As it is designed to work with chimeric spectra, Mango can be used on traditional high-resolution tandem mass spectrometry (MS/MS) capable mass spectrometers without the need for additional modifications. When paired with a traditional proteomics search engine, Mango can be used to identify several thousand cross-linked peptide pairs searching against the entire <i>Escherichia coli</i> proteome. Mango provides an avenue to perform whole proteome cross-linking experiments without specialized instrumentation or access to nonstandard methods

Large-Scale and Targeted Quantitative Cross-Linking MS Using Isotope-Labeled Protein Interaction Reporter (PIR) Cross-Linkers

Quantitative measurement of chemically cross-linked proteins is crucial to reveal dynamic information about protein structures and protein–protein interactions and how these are differentially regulated during stress, aging, drug treatment, and most perturbations. Previously, we demonstrated how quantitative in vivo cross-linking (CL) with stable isotope labeling of amino acids in cell culture (SILAC) enables both heritable and dynamic changes in cells to be visualized. In this work, we demonstrate the technical feasibility of proteome-scale quantitative in vivo CL–MS using isotope-labeled protein interaction reporter (PIR) cross-linkers and <i>E. coli</i> as a model system. This isotope-labeled cross-linkers approach, combined with Real-time Analysis of Cross-linked peptide Technology (ReACT) previously developed in our lab, enables the quantification of 941 nonredundant cross-linked peptide pairs from a total of 1213 fully identified peptide pairs in two biological replicate samples through comparison of MS¹ peak intensity of the light and heavy cross-linked peptide pairs. For targeted relative quantification of cross-linked peptide pairs, we further developed a PRM-based assay to accurately probe specific site interaction changes in a complex background. The methodology described in this work provides reliable tools for both large-scale and targeted quantitative CL–MS that is useful for any sample where SILAC labeling may not be practical

Cross-linking Measurements of the <i>Potato leafroll virus</i> Reveal Protein Interaction Topologies Required for Virion Stability, Aphid Transmission, and Virus–Plant Interactions

Protein interactions are critical determinants of insect transmission for viruses in the family <i>Luteoviridae</i>. Two luteovirid structural proteins, the capsid protein (CP) and the readthrough protein (RTP), contain multiple functional domains that regulate virus transmission. There is no structural information available for these economically important viruses. We used Protein Interaction Reporter (PIR) technology, a strategy that uses chemical cross-linking and high resolution mass spectrometry, to discover topological features of the <i>Potato leafroll virus</i> (PLRV) CP and RTP that are required for the diverse biological functions of PLRV virions. Four cross-linked sites were repeatedly detected, one linking CP monomers, two within the RTP, and one linking the RTP and CP. Virus mutants with triple amino acid deletions immediately adjacent to or encompassing the cross-linked sites were defective in virion stability, RTP incorporation into the capsid, and aphid transmission. Plants infected with a new, infectious PLRV mutant lacking 26 amino acids encompassing a cross-linked site in the RTP exhibited a delay in the appearance of systemic infection symptoms. PIR technology provided the first structural insights into luteoviruses which are crucially lacking and are involved in vector–virus and plant–virus interactions. These are the first cross-linking measurements on any infectious, insect-transmitted virus

Cross-linking Measurements of the <i>Potato leafroll virus</i> Reveal Protein Interaction Topologies Required for Virion Stability, Aphid Transmission, and Virus–Plant Interactions

Protein interactions are critical determinants of insect transmission for viruses in the family <i>Luteoviridae</i>. Two luteovirid structural proteins, the capsid protein (CP) and the readthrough protein (RTP), contain multiple functional domains that regulate virus transmission. There is no structural information available for these economically important viruses. We used Protein Interaction Reporter (PIR) technology, a strategy that uses chemical cross-linking and high resolution mass spectrometry, to discover topological features of the <i>Potato leafroll virus</i> (PLRV) CP and RTP that are required for the diverse biological functions of PLRV virions. Four cross-linked sites were repeatedly detected, one linking CP monomers, two within the RTP, and one linking the RTP and CP. Virus mutants with triple amino acid deletions immediately adjacent to or encompassing the cross-linked sites were defective in virion stability, RTP incorporation into the capsid, and aphid transmission. Plants infected with a new, infectious PLRV mutant lacking 26 amino acids encompassing a cross-linked site in the RTP exhibited a delay in the appearance of systemic infection symptoms. PIR technology provided the first structural insights into luteoviruses which are crucially lacking and are involved in vector–virus and plant–virus interactions. These are the first cross-linking measurements on any infectious, insect-transmitted virus

Cross-linking Measurements of the <i>Potato leafroll virus</i> Reveal Protein Interaction Topologies Required for Virion Stability, Aphid Transmission, and Virus–Plant Interactions

Protein interactions are critical determinants of insect transmission for viruses in the family <i>Luteoviridae</i>. Two luteovirid structural proteins, the capsid protein (CP) and the readthrough protein (RTP), contain multiple functional domains that regulate virus transmission. There is no structural information available for these economically important viruses. We used Protein Interaction Reporter (PIR) technology, a strategy that uses chemical cross-linking and high resolution mass spectrometry, to discover topological features of the <i>Potato leafroll virus</i> (PLRV) CP and RTP that are required for the diverse biological functions of PLRV virions. Four cross-linked sites were repeatedly detected, one linking CP monomers, two within the RTP, and one linking the RTP and CP. Virus mutants with triple amino acid deletions immediately adjacent to or encompassing the cross-linked sites were defective in virion stability, RTP incorporation into the capsid, and aphid transmission. Plants infected with a new, infectious PLRV mutant lacking 26 amino acids encompassing a cross-linked site in the RTP exhibited a delay in the appearance of systemic infection symptoms. PIR technology provided the first structural insights into luteoviruses which are crucially lacking and are involved in vector–virus and plant–virus interactions. These are the first cross-linking measurements on any infectious, insect-transmitted virus

Cross-linking Measurements of the <i>Potato leafroll virus</i> Reveal Protein Interaction Topologies Required for Virion Stability, Aphid Transmission, and Virus–Plant Interactions

Protein interactions are critical determinants of insect transmission for viruses in the family <i>Luteoviridae</i>. Two luteovirid structural proteins, the capsid protein (CP) and the readthrough protein (RTP), contain multiple functional domains that regulate virus transmission. There is no structural information available for these economically important viruses. We used Protein Interaction Reporter (PIR) technology, a strategy that uses chemical cross-linking and high resolution mass spectrometry, to discover topological features of the <i>Potato leafroll virus</i> (PLRV) CP and RTP that are required for the diverse biological functions of PLRV virions. Four cross-linked sites were repeatedly detected, one linking CP monomers, two within the RTP, and one linking the RTP and CP. Virus mutants with triple amino acid deletions immediately adjacent to or encompassing the cross-linked sites were defective in virion stability, RTP incorporation into the capsid, and aphid transmission. Plants infected with a new, infectious PLRV mutant lacking 26 amino acids encompassing a cross-linked site in the RTP exhibited a delay in the appearance of systemic infection symptoms. PIR technology provided the first structural insights into luteoviruses which are crucially lacking and are involved in vector–virus and plant–virus interactions. These are the first cross-linking measurements on any infectious, insect-transmitted virus

<i>In Vivo</i> Application of Photocleavable Protein Interaction Reporter Technology

<i>In vivo</i> protein structures and protein–protein interactions are critical to the function of proteins in biological systems. As a complementary approach to traditional protein interaction identification methods, cross-linking strategies are beginning to provide additional data on protein and protein complex topological features. Previously, photocleavable protein interaction reporter (pcPIR) technology was demonstrated by cross-linking pure proteins and protein complexes and the use of ultraviolet light to cleave or release cross-linked peptides to enable identification. In the present report, the pcPIR strategy is applied to <i>Escherichia coli</i> cells, and <i>in vivo</i> protein interactions and topologies are measured. More than 1600 labeled peptides from <i>E. coli</i> were identified, indicating that many protein sites react with pcPIR <i>in vivo</i>. From those labeled sites, 53 <i>in vivo</i> intercross-linked peptide pairs were identified and manually validated. Approximately half of the interactions have been reported using other techniques, although detailed structures exist for very few. Three proteins or protein complexes with detailed crystallography structures are compared to the cross-linking results obtained from <i>in vivo</i> application of pcPIR technology

Quantitative Proteomics Based on Optimized Data-Independent Acquisition in Plasma Analysis

The advent of high-resolution and frequency mass spectrometry has ushered in an era of data-independent acquisition (DIA). This approach affords enormous multiplexing capacity and is particularly suitable for clinical biomarker studies. However, DIA-based quantification of clinical plasma samples is a daunting task due to the high complexity of clinical plasma samples, the diversity of peptides within the samples, and the high biologic dynamic range of plasma proteins. Here we applied DIA methodology, including a highly reproducible sample preparation and LC–MS/MS analysis, and assessed its utility for clinical plasma biomarker detection. A pancreatic cancer-relevant plasma spectral library was constructed consisting of over 14 000 confidently identified peptides derived from over 2300 plasma proteins. Using a nonhuman protein as the internal standard, various empirical parameters were explored to maximize the reliability and reproducibility of the DIA quantification. The DIA parameters were optimized based on the quantification cycle times and fragmentation profile complexity. Higher analytical and biological reproducibility was recorded for the tryptic peptides without labile residues and missed cleavages. Quantification reliability was developed for the peptides identified within a consistent retention time and signal intensity. Linear analytical dynamic range and the lower limit of quantification were assessed, suggesting the critical role of sample complexity in optimizing DIA settings. Technical validation of the assay using a cohort of clinical plasma indicated the robustness and unique advantage for targeted analysis of clinical plasma samples in the context of biomarker development