Multiple and Sequential Data Acquisition Method: An Improved Method for Fragmentation and Detection of Cross-Linked Peptides on a Hybrid Linear Trap Quadrupole Orbitrap Velos Mass Spectrometer

The identification and validation of cross-linked peptides by mass spectrometry remains a daunting challenge for protein–protein cross-linking approaches when investigating protein interactions. This includes the fragmentation of cross-linked peptides in the mass spectrometer per se and following database searching, the matching of the molecular masses of the fragment ions to the correct cross-linked peptides. The hybrid linear trap quadrupole (LTQ) Orbitrap Velos combines the speed of the tandem mass spectrometry (MS/MS) duty circle with high mass accuracy, and these features were utilized in the current study to substantially improve the confidence in the identification of cross-linked peptides. An MS/MS method termed multiple and sequential data acquisition method (MSDAM) was developed. Preliminary optimization of the MS/MS settings was performed with a synthetic peptide (TP1) cross-linked with bis­[sulfosuccinimidyl] suberate (BS³). On the basis of these results, MSDAM was created and assessed on the BS³-cross-linked bovine serum albumin (BSA) homodimer. MSDAM applies a series of multiple sequential fragmentation events with a range of different normalized collision energies (NCE) to the same precursor ion. The combination of a series of NCE enabled a considerable improvement in the quality of the fragmentation spectra for cross-linked peptides, and ultimately aided in the identification of the sequences of the cross-linked peptides. Concurrently, MSDAM provides confirmatory evidence from the formation of reporter ions fragments, which reduces the false positive rate of incorrectly assigned cross-linked peptides

Statistical analysis of co-occurrence patterns in microbial presence-absence datasets - Fig 2

<p><b>Two examples of species pairs that are completely uncorrelated spatially that are incorrectly identified by the standard null model of Jaccard’s index</b> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187132#pone.0187132.ref006" target="_blank">6</a>] <b>as exhibiting negative (a) and positive (b) correlation.</b> Probability theory indicates that two events are independent if their joint probability is the product of marginal probabilities (also indicated by Chi square statistic). In agreement with probability theory, Veech’s null model for co-occurrence analysis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187132#pone.0187132.ref045" target="_blank">45</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187132#pone.0187132.ref056" target="_blank">56</a>] and our simulated, prevalence-specific null distribution place the observed <i>J</i> right at the center of the null distribution. However, the standard null model assigns an extremely low probability for the observed <i>J</i> given the null model, making it invalid for statistical inference of <i>J</i>.</p

Number of species pairs identified as significant by <i>J</i> and <i>r</i> as a function of species prevalence.

<p>The prevalence of two species in a given pair are shown on the two axes of the grids. After binning the prevalence at 5% interval, the total number of pairs significant in each grid cell was counted. Color scale across plots does not match; gray cell indicate lack of species pairs. Both <i>J</i> (a) and r (b) detect many species pairs significantly correlated (all positive) when at least one of the species in the pair is rare. However, when one of the species is abundant, unlike <i>J</i>, <i>r</i> fails to detect significant pairs (b). The difference in the number of species pairs significant for <i>J</i> and <i>r</i> shows a strong pattern with species prevalence (c). Total number of species pairs in the species prevalence grid is shown in (d). Of 844350 species pairs, 627539 (74.3%) have at least one of the species in the pair very rare (<10% prevalence) whereas 205120 (24.3%) have both species very rare.</p

A Comparative Proteomic Study of Human Skin Suction Blister Fluid from Healthy Individuals Using Immunodepletion and iTRAQ Labeling

Aberrations in skin morphology and functionality can cause acute and chronic skin-related diseases that are the focus of dermatological research. Mechanically induced skin suction blister fluid may serve as a potential, alternative human body fluid for quantitative mass spectrometry (MS)-based proteomics in order to assist in the understanding of the mechanisms and causes underlying skin-related diseases. The combination of abundant-protein removal with iTRAQ technology and multidimensional fractionation techniques improved the number of identified protein groups. A relative comparison of a cohort of 8 healthy volunteers was thus recruited in order to assess the net variability encountered in a healthy scenario. The technology enabled the identification, to date, of the highest number of reported protein groups (739) with concomitant relative quantitative data for over 90% of all proteins with high reproducibility and accuracy. The use of iTRAQ 8-plex resulted in a 66% decrease in protein identifications but, despite this, provided valuable insight into interindividual differences of the healthy control samples. The geometric mean ratio was close to 1 with 95% of all ratios ranging between 0.45 and 2.05 and a calculated mean coefficient of variation of 15.8%, indicating a lower biological variance than that reported for plasma or urine. By applying a multistep sample processing, the obtained sensitivity and accuracy of quantitative MS analysis demonstrates the prospective value of the approach in future research into skin diseases

A Comparative Proteomic Study of Human Skin Suction Blister Fluid from Healthy Individuals Using Immunodepletion and iTRAQ Labeling

Aberrations in skin morphology and functionality can cause acute and chronic skin-related diseases that are the focus of dermatological research. Mechanically induced skin suction blister fluid may serve as a potential, alternative human body fluid for quantitative mass spectrometry (MS)-based proteomics in order to assist in the understanding of the mechanisms and causes underlying skin-related diseases. The combination of abundant-protein removal with iTRAQ technology and multidimensional fractionation techniques improved the number of identified protein groups. A relative comparison of a cohort of 8 healthy volunteers was thus recruited in order to assess the net variability encountered in a healthy scenario. The technology enabled the identification, to date, of the highest number of reported protein groups (739) with concomitant relative quantitative data for over 90% of all proteins with high reproducibility and accuracy. The use of iTRAQ 8-plex resulted in a 66% decrease in protein identifications but, despite this, provided valuable insight into interindividual differences of the healthy control samples. The geometric mean ratio was close to 1 with 95% of all ratios ranging between 0.45 and 2.05 and a calculated mean coefficient of variation of 15.8%, indicating a lower biological variance than that reported for plasma or urine. By applying a multistep sample processing, the obtained sensitivity and accuracy of quantitative MS analysis demonstrates the prospective value of the approach in future research into skin diseases

A Comparative Proteomic Study of Human Skin Suction Blister Fluid from Healthy Individuals Using Immunodepletion and iTRAQ Labeling

Aberrations in skin morphology and functionality can cause acute and chronic skin-related diseases that are the focus of dermatological research. Mechanically induced skin suction blister fluid may serve as a potential, alternative human body fluid for quantitative mass spectrometry (MS)-based proteomics in order to assist in the understanding of the mechanisms and causes underlying skin-related diseases. The combination of abundant-protein removal with iTRAQ technology and multidimensional fractionation techniques improved the number of identified protein groups. A relative comparison of a cohort of 8 healthy volunteers was thus recruited in order to assess the net variability encountered in a healthy scenario. The technology enabled the identification, to date, of the highest number of reported protein groups (739) with concomitant relative quantitative data for over 90% of all proteins with high reproducibility and accuracy. The use of iTRAQ 8-plex resulted in a 66% decrease in protein identifications but, despite this, provided valuable insight into interindividual differences of the healthy control samples. The geometric mean ratio was close to 1 with 95% of all ratios ranging between 0.45 and 2.05 and a calculated mean coefficient of variation of 15.8%, indicating a lower biological variance than that reported for plasma or urine. By applying a multistep sample processing, the obtained sensitivity and accuracy of quantitative MS analysis demonstrates the prospective value of the approach in future research into skin diseases

A Comparative Proteomic Study of Human Skin Suction Blister Fluid from Healthy Individuals Using Immunodepletion and iTRAQ Labeling

Aberrations in skin morphology and functionality can cause acute and chronic skin-related diseases that are the focus of dermatological research. Mechanically induced skin suction blister fluid may serve as a potential, alternative human body fluid for quantitative mass spectrometry (MS)-based proteomics in order to assist in the understanding of the mechanisms and causes underlying skin-related diseases. The combination of abundant-protein removal with iTRAQ technology and multidimensional fractionation techniques improved the number of identified protein groups. A relative comparison of a cohort of 8 healthy volunteers was thus recruited in order to assess the net variability encountered in a healthy scenario. The technology enabled the identification, to date, of the highest number of reported protein groups (739) with concomitant relative quantitative data for over 90% of all proteins with high reproducibility and accuracy. The use of iTRAQ 8-plex resulted in a 66% decrease in protein identifications but, despite this, provided valuable insight into interindividual differences of the healthy control samples. The geometric mean ratio was close to 1 with 95% of all ratios ranging between 0.45 and 2.05 and a calculated mean coefficient of variation of 15.8%, indicating a lower biological variance than that reported for plasma or urine. By applying a multistep sample processing, the obtained sensitivity and accuracy of quantitative MS analysis demonstrates the prospective value of the approach in future research into skin diseases

A Comparative Proteomic Study of Human Skin Suction Blister Fluid from Healthy Individuals Using Immunodepletion and iTRAQ Labeling

Aberrations in skin morphology and functionality can cause acute and chronic skin-related diseases that are the focus of dermatological research. Mechanically induced skin suction blister fluid may serve as a potential, alternative human body fluid for quantitative mass spectrometry (MS)-based proteomics in order to assist in the understanding of the mechanisms and causes underlying skin-related diseases. The combination of abundant-protein removal with iTRAQ technology and multidimensional fractionation techniques improved the number of identified protein groups. A relative comparison of a cohort of 8 healthy volunteers was thus recruited in order to assess the net variability encountered in a healthy scenario. The technology enabled the identification, to date, of the highest number of reported protein groups (739) with concomitant relative quantitative data for over 90% of all proteins with high reproducibility and accuracy. The use of iTRAQ 8-plex resulted in a 66% decrease in protein identifications but, despite this, provided valuable insight into interindividual differences of the healthy control samples. The geometric mean ratio was close to 1 with 95% of all ratios ranging between 0.45 and 2.05 and a calculated mean coefficient of variation of 15.8%, indicating a lower biological variance than that reported for plasma or urine. By applying a multistep sample processing, the obtained sensitivity and accuracy of quantitative MS analysis demonstrates the prospective value of the approach in future research into skin diseases

Comparison of co-absent site percentages from different macroecological studies and our current microbiome study.

<p>Comparison of co-absent site percentages from different macroecological studies and our current microbiome study.</p

Examples of studies that used presence-absence data to compute Jaccard’s similarity index (<i>J</i>) for determining similarity between systems (e.g., between taxa-pairs, between sites, between markets) where the statistical significance of <i>J</i> is faulty and the use of observed value of <i>J</i> as a similarity metric is flawed.

<p>Examples of studies that used presence-absence data to compute Jaccard’s similarity index (<i>J</i>) for determining similarity between systems (e.g., between taxa-pairs, between sites, between markets) where the statistical significance of <i>J</i> is faulty and the use of observed value of <i>J</i> as a similarity metric is flawed.</p