Label-free peptide profiling of Orbitrap™ full mass spectra

Background. We developed a new version of the open source software package Peptrix that can yet compare large numbers of Orbitrap™ LC-MS data. The peptide profiling results for Peptrix on MS1 spectra were compared with those obtained from a small selection of open source and commercial software packages: msInspect, Sieve™ and Progenesis™. The properties compared in these packages were speed, total number of detected masses, redundancy of masses, reproducibility in numbers and CV of intensity, overlap of masses, and differences in peptide peak intensities. Reproducibility measurements were taken for the different MS1 software applications by measuring in triplicate a complex peptide mixture of immunoglobulin on the Orbitrap™ mass spectrometer. Values of peptide masses detected from the high intensity peaks of the MS1 spectra by peptide profiling were verified with values of the MS2 fragmented and sequenced masses that resulted in protein identifications with a significant score. Findings. Peptrix finds about the same number of peptide features as the other packages, but peptide masses are in some cases approximately 5 to 10 times less redundant present in the peptide profile matrix. The Peptrix profile matrix displays the largest overlap when comparing the number of masses in a pair between two software applications. The overlap of peptide masses between software packages of low intensity peaks in the spectra is remarkably low with about 50% of the detected masses in the individual packages. Peptrix does not differ from the other packages in detecting 96% of the masses that relate to highly abundant sequenced proteins. MS1 peak intensities vary between the applications in a non linear way as they are not processed using the same method. Conclusions. Peptrix is capable of peptide profiling using Orbitrap™ files and finding differential expressed peptides in body fluid and tissue samples. The number of peptide masses detected in Orbitrap™ files can be increased by using more MS1 peptide profiling applications, including Peptrix, since it appears from the comparison of Peptrix with the other applications that all software packages have likely a high false negative rate of low intensity peptide peaks (missing peptides)

De Novo Sequencing of Peptides from High-Resolution Bottom-Up Tandem Mass Spectra using Top-Down Intended Methods

Despite high-resolution mass spectrometers are becoming accessible for more and more laboratories, tandem (MS/MS) mass spectra are still often collected at a low resolution. And even if acquired at a high resolution, software tools used for their processing do not tend to benefit from that in full, and an ability to specify a relative mass tolerance in this case often remains the only feature the respective algorithms take advantage of. We argue that a more efficient way to analyze high-resolution MS/MS spectra should be with methods more explicitly accounting for the precision level, and sustain this claim through demonstrating that a de novo sequencing framework originally developed for (high-resolution) top-down MS/MS data is perfectly suitable for processing high-resolution bottom-up datasets, even though a top-down like deconvolution performed as the first step will leave in many spectra at most a few peaks

Top-down analysis of protein samples by de novo sequencing techniques

Motivation: Recent technological advances have made high-resolution mass spectrometers affordable to many laboratories, thus boosting rapid development of top-down mass spectrometry, and implying a need in efficient methods for analyzing this kind of data. Results: We describe a method for analysis of protein samples from top-down tandem mass spectrometry data, which capitalizes on de novo sequencing of fragments of the proteins present in the sample. Our algorithm takes as input a set of de novo amino acid strings derived from the given mass spectra using the recently proposed Twister approach, and combines them into aggregated strings endowed with offsets. The former typically constitute accurate sequence fragments of sufficiently well-represented proteins from the sample being analyzed, while the latter indicate their location in the protein sequence, and also bear information on post-translational modifications and fragmentation patterns. Availability and Implementation: Freely available on the web at http://bioinf.spbau.ru/en/twister

A database application for pre-processing, storage and comparison of mass spectra derived from patients and controls.

BACKGROUND: Statistical comparison of peptide profiles in biomarker discovery requires fast, user-friendly software for high throughput data analysis. Important features are flexibility in changing input variables and statistical analysis of peptides that are differentially expressed between patient and control groups. In addition, integration the mass spectrometry data with the results of other experiments, such as microarray analysis, and information from other databases requires a central storage of the profile matrix, where protein id's can be added to peptide masses of interest. RESULTS: A new database application is presented, to detect and identify significantly differentially expressed peptides in peptide profiles obtained from body fluids of patient and control groups. The presented modular software is capable of central storage of mass spectra and results in fast analysis. The software architecture consists of 4 pillars, 1) a Graphical User Interface written in Java, 2) a MySQL database, which contains all metadata, such as experiment numbers and sample codes, 3) a FTP (File Transport Protocol) server to store all raw mass spectrometry files and processed data, and 4) the software package R, which is used for modular statistical calculations, such as the Wilcoxon-Mann-Whitney rank sum test. Statistic analysis by the Wilcoxon-Mann-Whitney test in R demonstrates that peptide-profiles of two patient groups 1) breast cancer patients with leptomeningeal metastases and 2) prostate cancer patients in end stage disease can be distinguished from those of control groups. CONCLUSION: The database application is capable to distinguish patient Matrix Assisted Laser Desorption Ionization (MALDI-TOF) peptide profiles from control groups using large size datasets. The modular architecture of the application makes it possible to adapt the application to handle also large sized data from MS/MS- and Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass spectrometry experiments. It is expected that the higher resolution and mass accuracy of the FT-ICR mass spectrometry prevents the clustering of peaks of different peptides and allows the identification of differentially expressed proteins from the peptide profiles

Ultra-Fast Analysis of Plasma and Intracellular Levels of HIV Protease Inhibitors in Children: A Clinical Application of MALDI Mass Spectrometry

HIV protease inhibitors must penetrate into cells to exert their action. Differences in the intracellular pharmacokinetics of these drugs may explain why some patients fail on therapy or suffer from drug toxicity. Yet, there is no information available on the intracellular levels of HIV protease inhibitors in HIV infected children, which is in part due to the large amount of sample that is normally required to measure the intracellular concentrations of these drugs. Therefore, we developed an ultra-fast and sensitive assay to measure the intracellular concentrations of HIV protease inhibitors in small amounts of peripheral blood mononuclear cells (PBMCs), and determined the intracellular concentrations of lopinavir and ritonavir in HIV infected children. An assay based on matrix-assisted laser desorption/ionization (MALDI) - triple quadrupole mass spectrometry was developed to determine the concentrations of HIV protease inhibitors in 10 µL plasma and 1×106 PBMCs. Precisions and accuracies were within the values set by the FDA for bioanalytical method validation. Lopinavir and ritonavir did not accumulate in PBMCs of HIV infected children. In addition, the intracellular concentrations of lopinavir and ritonavir correlated poorly to the plasma concentrations of these drugs. MALDI-triple quadrupole mass spectrometry is a new tool for ultra-fast and sensitive determination of drug concentrations which can be used, for example, to assess the intracellular pharmacokinetics of HIV protease inhibitors in HIV infected children

Comparison of Targeted Mass Spectrometry Techniques With an Immunoassay:A Case Study For HSP90α

PURPOSE: The objective of this study was to better understand factors governing the variability and sensitivity in SRM and PRM, compared to immunoassay. EXPERIMENTAL DESIGN: A 2D-LC-MS/MS-based SRM and PRM assay was developed for quantitative measurements of HSP90α in serum. Forty-three control sera were compared by SRM, PRM and ELISA following the manufacturer's instructions. Serum samples were trypsin-digested and fractionated by SCX chromatography prior to SRM and PRM measurements. Analytical parameters such as linearity, LOD, LOQ, repeatability and reproducibility of the SRM, PRM and ELISA were determined. RESULTS: PRM data obtained by high-resolution mass spectrometry correlated better with ELISA measurements than SRM data measured on a triple quadrupole mass spectrometer. While all three methods (SRM, PRM, ELISA) were able to quantify HSP90α in serum at the ng/mL level, the use of PRM on a high-resolution mass spectrometer reduced variation and showed comparable sensitivity to immunoassay. CONCLUSIONS AND CLINICAL RELEVANCE: Using fractionation, it is possible to measure ng/mL levels of HSP90α in a reproducible, selective and sensitive way using PRM in serum. This opens up the possibility to use PRM in a multiplexed way as an attractive alternative for immunoassays without the use of antibodies or comparable binders. This article is protected by copyright. All rights reserved

TP53 mutated glioblastoma stem-like cell cultures are sensitive to dual mTORC1/2 inhibition while resistance in TP53 wild type cultures can be overcome by combined inhibition of mTORC1/2 and Bcl-2

Background: Glioblastoma is the most malignant tumor of the central nervous system and still lacks effective treatment. This study explores mutational biomarkers of 11 drugs targeting either the RTK/Ras/PI3K, the p53 or the Rb pathway using 25 patient-derived glioblastoma stem-like cell cultures (GSCs). Results: We found that TP53 mutated GSCs were approximately 3.5 fold more sensitive to dual inhibition of mammalian target of rapamycin complex 1 and 2 (mTORC1/2) compared to wild type GSCs. We identified that Bcl-2(Thr56/Ser70) phosphorylation contributed to the resistance of TP53 wild type GSCs against dual mTORC1/2 inhibition. The Bcl-2 inhibitor ABT-263 (navitoclax) increased sensitivity to the mTORC1/2 inhibitor AZD8055 in TP53 wild type GSCs, while sensitivity to AZD8055 in TP53 mutated GSCs remained unchanged. Conclusion: Our data suggest that Bcl-2 confers resistance to mTORC1/2 inhibitors in TP53 wild type GSCs and that combined inhibition of both mTORC1/2 and Bcl-2 is worthwhile to explore further in TP53 wild type glioblastomas, whereas in TP53 mutated glioblastomas dual mTORC1/2 inhibitors should be explored

An antibody-based biomarker discovery method by mass spectrometry sequencing of complementarity determining regions

Autoantibodies are increasingly used as biomarkers in the detection of autoimmune disorders and cancer. Disease specific antibodies are generally detected by their binding to specific antigens. As an alternative approach, we propose to identify specific complementarity determining regions (CDR) of IgG that relate to an autoimmune disorder or cancer instead of the specific antigen(s). In this manuscript, we tested the technical feasibility to detect and identify CDRs of specific antibodies by mass spectrometry. We used a commercial pooled IgG preparation as well as purified serum IgG fractions that were spiked with different amounts of a fully human monoclonal antibody (adalimumab). These samples were enzymatically digested and analyzed by nanoLC Orbitrap mass spectrometry. In these samples, we were able to identify peptides derived from the CDRs of adalimumab. These peptides could be detected at an amount of 110 attomole, 5 orders of magnitude lower than the total IgG concentration in these samples. Using higher energy collision induced dissociation (HCD) fragmentation and subsequent de novo sequencing, we could successfully identify 50% of the detectable CDR peptides of adalimumab. In addition, we demonstrated that an affinity purification with anti-dinitrophenol (DNP) monoclonal antibody enhanced anti-DNP derived CDR detection in a serum IgG background. In conclusion, specific CDR peptides could be detected and sequenced at relatively low levels (attomole-femtomole range) which should allow the detection of clinically relevant CDR peptides in patient samples