173 research outputs found
Separate & analyze:Improved mass spectrometry-based clinical proteomics by fractionation
The proteome of clinical specimens is complex, and its entirety cannot be fully captured by current methods. By separating the proteome into fractions, it becomes possible to analyze it in greater depth, i.e. to identify and quantify proteins that are present in relatively low amounts. The separation of the proteome and the analytic disassembly into its components can be performed in various ways at different stages of an analysis. This thesis describes the realization of clinical proteomic studies using analytical methods for separation and fractionation of cells, proteins, and peptides. In Chapter 2, we sampled isolated populations of epithelial and stromal cells in non-dysplastic and dysplastic/carcinogenic tissue samples taken from the esophagus of patients with different stages of the dysplastic progression from Barrettâs esophagus to esophageal adenocarcinoma. This approach allowed us to determine proteomic alterations in dysplastic/cancerous cell compartments, which quantitatively represent only a small proportion of the entire tissue specimen. Chapters 3 and 4 address biomarker discovery and validation in cerebrospinal fluid (CSF) using mass spectrometry-based proteomics. In preceding stages, we investigated the applicability of two different separation techniques. In Chapter 3, we removed albumin and immunoglobulins (Ig) from CSF through immunoaffinity depletion and conducted a label-free quantitative proteomic discovery study on depleted CSF fractions. In Chapter 4, we conducted a preceding pilot study to explore the use of two-dimensional (2D) chromatography to increase proteome coverage and depth in CSF samples. In Chapter 5, we applied 2D chromatography and ion mobility gas-phase fractionation to improve the detection of antibody variable region peptides. Chapter 6 involved the assessment of the phosphoproteome in fresh-frozen and FFPE brain tissue samples using IMAC phosphopeptide enrichment. The work of Chapter 6 was adapted and extended in Chapter 7, using the fraction of phosphopeptides as an epitope substrate for serum antibodies targeting tumor-specific phosphosites. The antibody-peptide binding assay presented in this chapter indicated the presence of a serum antibody against a glioblastoma multiforme-associated phosphosite. In Chapter 8, we introduced a data processing method for the analysis of quantitative LC-MS data. Parallel reaction monitoring (PRM) is a common and versatile quantitative MS technique, which was applied in Chapters 3, 4, 6, and 7. The tool presented in Chapter 8 enhances reproducibility, confidence, and speed during data analysis.<br/
Separate & analyze:Improved mass spectrometry-based clinical proteomics by fractionation
The proteome of clinical specimens is complex, and its entirety cannot be fully captured by current methods. By separating the proteome into fractions, it becomes possible to analyze it in greater depth, i.e. to identify and quantify proteins that are present in relatively low amounts. The separation of the proteome and the analytic disassembly into its components can be performed in various ways at different stages of an analysis. This thesis describes the realization of clinical proteomic studies using analytical methods for separation and fractionation of cells, proteins, and peptides. In Chapter 2, we sampled isolated populations of epithelial and stromal cells in non-dysplastic and dysplastic/carcinogenic tissue samples taken from the esophagus of patients with different stages of the dysplastic progression from Barrettâs esophagus to esophageal adenocarcinoma. This approach allowed us to determine proteomic alterations in dysplastic/cancerous cell compartments, which quantitatively represent only a small proportion of the entire tissue specimen. Chapters 3 and 4 address biomarker discovery and validation in cerebrospinal fluid (CSF) using mass spectrometry-based proteomics. In preceding stages, we investigated the applicability of two different separation techniques. In Chapter 3, we removed albumin and immunoglobulins (Ig) from CSF through immunoaffinity depletion and conducted a label-free quantitative proteomic discovery study on depleted CSF fractions. In Chapter 4, we conducted a preceding pilot study to explore the use of two-dimensional (2D) chromatography to increase proteome coverage and depth in CSF samples. In Chapter 5, we applied 2D chromatography and ion mobility gas-phase fractionation to improve the detection of antibody variable region peptides. Chapter 6 involved the assessment of the phosphoproteome in fresh-frozen and FFPE brain tissue samples using IMAC phosphopeptide enrichment. The work of Chapter 6 was adapted and extended in Chapter 7, using the fraction of phosphopeptides as an epitope substrate for serum antibodies targeting tumor-specific phosphosites. The antibody-peptide binding assay presented in this chapter indicated the presence of a serum antibody against a glioblastoma multiforme-associated phosphosite. In Chapter 8, we introduced a data processing method for the analysis of quantitative LC-MS data. Parallel reaction monitoring (PRM) is a common and versatile quantitative MS technique, which was applied in Chapters 3, 4, 6, and 7. The tool presented in Chapter 8 enhances reproducibility, confidence, and speed during data analysis.<br/
Cj0683 Is a Competence Protein Essential for Efficient Initialization of DNA Uptake in Campylobacter jejuni
C. jejuni is an important food-borne pathogen displaying high genetic diversity, substantially based on natural transformation. The mechanism of DNA uptake from the environment depends on a type II secretion/type IV pilus system, whose components are partially known. Here, we quantified DNA uptake in C. jejuni at the single cell level and observed median transport capacities of approximately 30 kb per uptake location. The process appeared to be limited by the initialization of DNA uptake, was finite, and, finalized within 30 min of contact to DNA. Mutants lacking either the outer membrane pore PilQ or the inner membrane channel ComEC were deficient in natural transformation. The periplasmic DNA binding protein ComE was negligible for DNA uptake, which is in contrast to its proposed function. Intriguingly, a mutant lacking the unique periplasmic protein Cj0683 displayed rare but fully functional DNA uptake events. We conclude that Cj0683 was essential for the efficient initialization of DNA uptake, consistent with the putative function as a competence pilus protein. Unravelling features important in natural transformation might lead to target identification, reducing the adaptive potential of pathogens
Improved detection of tryptic immunoglobulin variable region peptides by chromatographic and gas-phase fractionation techniques
The polyclonal repertoire of circulating antibodies potentially holds valuable information about an individual's humoral immune state. While bottom-up proteomics is well suited for serum proteomics, the vast number of antibodies and dynamic range of serum challenge this analysis. To acquire the serum proteome more comprehensively, we incorporated high-field asymmetric waveform ion-mobility spectrometry (FAIMS) or two-dimensional chromatography into standard trypsin-based bottom-up proteomics. Thereby, the number of variable region (VR)-related spectra increased 1.7-fold with FAIMS and 10-fold with chromatography fractionation. To match antibody VRs to spectra, we combined de novo searching and BLAST alignment. Validation of this approach showed that, as peptide length increased, the de novo accuracy decreased and BLAST performance increased. Through in silico calculations on antibody repository sequences, we determined the uniqueness of tryptic VR peptides and their suitability as antibody surrogate. Approximately one-third of these peptides were unique, and about one-third of all antibodies contained at least one unique peptide.</p
Improved detection of tryptic immunoglobulin variable region peptides by chromatographic and gas-phase fractionation techniques
The polyclonal repertoire of circulating antibodies potentially holds valuable information about an individual's humoral immune state. While bottom-up proteomics is well suited for serum proteomics, the vast number of antibodies and dynamic range of serum challenge this analysis. To acquire the serum proteome more comprehensively, we incorporated high-field asymmetric waveform ion-mobility spectrometry (FAIMS) or two-dimensional chromatography into standard trypsin-based bottom-up proteomics. Thereby, the number of variable region (VR)-related spectra increased 1.7-fold with FAIMS and 10-fold with chromatography fractionation. To match antibody VRs to spectra, we combined de novo searching and BLAST alignment. Validation of this approach showed that, as peptide length increased, the de novo accuracy decreased and BLAST performance increased. Through in silico calculations on antibody repository sequences, we determined the uniqueness of tryptic VR peptides and their suitability as antibody surrogate. Approximately one-third of these peptides were unique, and about one-third of all antibodies contained at least one unique peptide.</p
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 Progenesisâ˘. 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)
Proteomic characterization of microdissected breast tissue environment provides a protein-level overview of malignant transformation
Both healthy and cancerous breast tissue is heterogeneous, which is a bottleneck for proteomicsâbased biomarker analysis, as it obscures the cellular origin of a measured protein. We therefore aimed at obtaining a proteinâlevel interpretation of malignant transformation through global proteome analysis of a variety of laser capture microdissected cells originating from benign and malignant breast tissues. We compared proteomic differences between these tissues, both from cells of epithelial origin and the stromal environment, and performed string analysis. Differences in protein abundances corresponded with several hallmarks of cancer, including loss of cell adhesion, transformation to a migratory phenotype, and enhanced energy metabolism. Furthermore, despite enriching for (tumor) epithelial cells, many changes to the extracellular matrix were detected in microdissected cells of epithelial origin. The stromal compartment was heterogeneous and richer in the number of fibroblast and immune cells in malignant sections, compared to benign tissue sections. Furthermore, stroma could be clearly divided into reactive and nonreactive based on extracellular matrix disassembly proteins. We conclude that proteomics analysis of both microdissected epithelium and stroma gives an additional layer of information and more detailed insight into malignant transformation
Structure and Function of the Campylobacter jejuni Chromosome Replication Origin
Campylobacter jejuni is the leading bacterial cause of foodborne infections worldwide. However, our understanding of its cell cycle is poor. We identified the probable C. jejuni origin of replication (oriC) â a key element for initiation of chromosome replication, which is also important for chromosome structure, maintenance and dynamics. The herein characterized C. jejuni oriC is monopartite and contains (i) the DnaA box cluster, (ii) the DnaA-dependent DNA unwinding element (DUE) and (iii) binding sites for regulatory proteins. The cluster of five DnaA boxes and the DUE were found in the dnaA-dnaN intergenic region. Binding of DnaA to this cluster of DnaA-boxes enabled unwinding of the DUE in vitro. However, it was not sufficient to sustain replication of minichromosomes, unless the cluster was extended by additional DnaA boxes located in the 3Ⲡend of dnaA. This suggests, that C. jejuni oriC requires these boxes to initiate or to regulate replication of its chromosome. However, further detailed mutagenesis is required to confirm the role of these two boxes in initiation of C. jejuni chromosome replication and thus to confirm partial localization of C. jejuni oriC within a coding region, which has not been reported thus far for any bacterial oriC. In vitro DUE unwinding by DnaA was inhibited by Cj1509, an orphan response regulator and a homolog of HP1021, that has been previously shown to inhibit replication in Helicobacter pylori. Thus, Cj1509 might play a similar role as a regulator of C. jejuni chromosome replication. This is the first systematic analysis of chromosome replication initiation in C. jejuni, and we expect that these studies will provide a basis for future research examining the structure and dynamics of the C. jejuni chromosome, which will be crucial for understanding the pathogensâ life cycle and virulence
Proteomic alterations in early stage cervical cancer
Laser capture microdissection (LCM) allows the capture of cell types or well-defined structures in tissue. We compared in a semi-quantitative way the proteomes from an equivalent of 8,000 tumor cells from patients with squamous cell cervical cancer (SCC, n = 22) with healthy epithelial and stromal cells obtained from normal cervical tissue (n = 13). Proteins were enzymatically digested into peptides which were measured by high-resolution mass spectrometry and analyzed by âall-or-nothingâ analysis, Bonferroni, and Benjamini-Hochberg correction for multiple testing. By comparing LCM cell type preparations, 31 proteins were exclusively found in early stage cervical cancer (n = 11) when compared with healthy epithelium and stroma, based on criteria that address specificity in a restrictive âall-or-nothingâ way. By Bonferroni correction for multiple testing, 30 proteins were significantly up-regulated between early stage cervical cancer and healthy control, including six members of the MCM protein family. MCM proteins are involved in DNA repair and expected to be participating in the early stage of cancer. After a less stringent Benjamini-Hochberg correction for multiple testing, we found that the abundances of 319 proteins were significantly different between early stage cervical cancer and healthy controls. Four proteins were confirmed in digests of whole tissue lysates by Parallel Reaction Monitoring (PRM). Ingenuity Pathway Analysis using correction for multiple testing by permutation resulted in two networks that were differentially regulated in early stage cervical cancer compared with healthy tissue. From these networks, we learned that specific tumor mechanisms become effective during the early stage of cervical cancer
SIMPATIQCO: A server-based software suite which facilitates monitoring the time course of LC-MS performance metrics on orbitrap instruments
While the performance of liquid chromatography (LC) and mass spectrometry (MS) instrumentation continues to increase, applications such as analyses of complete or near-complete proteomes and quantitative studies require constant and optimal system performance. For this reason, research laboratories and core facilities alike are recommended to implement quality control (QC) measures as part of their routine workflows. Many laboratories perform sporadic quality control checks. However, successive and systematic longitudinal monitoring of system performance would be facilitated by dedicated automatic or semiautomatic software solutions that aid an effortless analysis and display of QC metrics over time. We present the software package SIMPATIQCO (SIMPle AuTomatIc Quality COntrol) designed for evaluation of data from LTQ Orbitrap, Q-Exactive, LTQ FT, and LTQ instruments. A centralized SIMPATIQCO server can process QC data from multiple instruments. The software calculates QC metrics supervising every step of data acquisition from LC and electrospray to MS. For each QC metric the software learns the range indicating adequate system performance from the uploaded data using robust statistics. Results are stored in a database and can be displayed in a comfortable manner from any computer in the laboratory via a web browser. QC data can be monitored for individual LC runs as well as plotted over time. SIMPATIQCO thus assists the longitudinal monitoring of important QC metrics such as peptide elution times, peak widths, intensities, total ion current (TIC) as well as sensitivity, and overall LC-MS system performance; in this way the software also helps identify potential problems. The SIMPATIQCO software package is available free of charge
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