laser-microdissection-based-protocol-for-lc-msms-analysis-proteomic

Laser Microdissection-Based Protocol for the LC-MS/MS Analysis of the Proteomic Profile of Neuromelanin Granules

Neuromelanin is a black-brownish pigment, present in so-called neuromelanin granules (NMGs) in dopaminergic neurons of the substantia nigra pars compacta. ...

Ruhr-University Bochum

In proteomics, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is established for identifying peptides and proteins. Duplicated spectra, that is, multiple spectra of the same peptide, occur both in single MS/MS runs and in large spectral libraries. Clustering tandem mass spectra is used to find consensus spectra, with manifold applications. First, it speeds up database searches, as performed for instance by Mascot. Second, it helps to identify novel peptides across species. Third, it is used for quality control to detect wrongly annotated spectra. We compare different clustering algorithms based on the cosine distance between spectra. CAST, MS-Cluster, and PRIDE Cluster are popular algorithms to cluster tandem mass spectra. We add well-known algorithms for large data sets, hierarchical clustering, DBSCAN, and connected components of a graph, as well as the new method N-Cluster. All algorithms are evaluated on real data with varied parameter settings. Cluster results are compared with each other and with peptide annotations based on validation measures such as purity. Quality control, regarding the detection of wrongly (un)annotated spectra, is discussed for exemplary resulting clusters. N-Cluster proves to be highly competitive. All clustering results benefit from the so-called DISMS2 filter that integrates additional information, for example, on precursor mass.

Comparison and Evaluation of Clustering Algorithms for Tandem Mass Spectra.

Cerebrospinal fluid is investigated in biomarker studies for various neurological disorders of the central nervous system due to its proximity to the brain. Currently, only a limited number of biomarkers have been validated in independent studies. The high variability in the protein composition and protein abundance of cerebrospinal fluid between as well as within individuals might be an important reason for this phenomenon. To evaluate this possibility, we investigated the inter- and intraindividual variability in the cerebrospinal fluid proteome globally, with a specific focus on disease biomarkers described in the literature. Cerebrospinal fluid from a longitudinal study group including 12 healthy control subjects was analyzed by label-free quantification (LFQ) via LC-MS/MS. Data were quantified via MaxQuant. Then, the intra- and interindividual variability and the reference change value were calculated for every protein. We identified and quantified 791 proteins, and 216 of these proteins were abundant in all samples and were selected for further analysis. For these proteins, we found an interindividual coefficient of variation of up to 101.5% and an intraindividual coefficient of variation of up to 29.3%. Remarkably, these values were comparably high for both proteins that were published as disease biomarkers and other proteins. Our results support the hypothesis that natural variability greatly impacts cerebrospinal fluid protein biomarkers because high variability can lead to unreliable results. Thus, we suggest controlling the variability of each protein to distinguish between good and bad biomarker candidates, e.g., by utilizing reference change values to improve the process of evaluating potential biomarkers in future studies.

Protein variability in cerebrospinal fluid and its possible implications for neurological protein biomarker research.

Histopathological differentiation between severe urocystitis with reactive urothelial atypia and carcinoma in situ (CIS) can be difficult, particularly after a treatment that deliberately induces an inflammatory reaction, such as intravesical instillation of Bacillus Calmette-Guèrin. However, precise grading in bladder cancer is critical for therapeutic decision making and thus requires reliable immunohistochemical biomarkers. Herein, an exemplary potential biomarker in bladder cancer was identified by the novel approach of Fourier transform infrared imaging for label-free tissue annotation of tissue thin sections. Identified regions of interest are collected by laser microdissection to provide homogeneous samples for liquid chromatography-tandem mass spectrometry-based proteomic analysis. This approach afforded label-free spatial classification with a high accuracy and without interobserver variability, along with the molecular resolution of the proteomic analysis. Cystitis and invasive high-grade urothelial carcinoma samples were analyzed. Three candidate biomarkers were identified and verified by immunohistochemistry in a small cohort, including low-grade urothelial carcinoma samples. The best-performing candidate AHNAK2 was further evaluated in a much larger independent verification cohort that also included CIS samples. Reactive urothelial atypia and CIS were distinguishable on the basis of the expression of this newly identified and verified immunohistochemical biomarker, with a sensitivity of 97% and a specificity of 69%. AHNAK2 can differentiate between reactive urothelial atypia in the setting of an acute or chronic cystitis and nonmuscle invasive-type CIS.

Integrated Fourier Transform Infrared Imaging and Proteomics for Identification of a Candidate Histochemical Biomarker in Bladder Cancer.

Acute myeloid leukemia (AML) is, if untreated, a fatal hematologic neoplasia. Failure of the first induction chemotherapy is a hallmark for a poor prognosis. Early recognition of therapy failure is crucial for planning further therapies. Therefore, international guidelines recommend a bone marrow biopsy around day 14 after the beginning of induction therapy. Hypocellular bone marrow on day 14 is still gold standard for therapy assessment and further therapy strategy. Despite this, non-invasive ways for the evaluation of induction therapy were looked for in the past years.

Monocytes-neutrophils-ratio as predictive marker for failure of first induction therapy in AML.

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a curative approach for multiple hematologic diseases. The success of alloHSCT is evaluated by analyzing the proportion of living donor cells in blood and bone marrow samples of the recipient (chimerism analysis). To monitor the engrafted cells, donor's individual genetic markers are analyzed in peripheral blood and bone marrow samples, usually by using short tandem repeat (STR) analysis. An alternative method to measure chimerism is based on insertion and deletion markers (InDels) analyzed by digital droplet PCR (ddPCR); however, this approach is rarely evaluated in clinical practice.

Digital droplet PCR-based chimerism analysis for monitoring of hematopoietic engraftment after allogeneic stem cell transplantation.

To investigate the effect of therapeutic suggestions played to patients through earphones during surgery on postoperative pain and opioid use.

Effect of therapeutic suggestions during general anaesthesia on postoperative pain and opioid use: multicentre randomised controlled trial.

This article describes some use case studies and self-assessments of FAIR status of de.NBI services to illustrate the challenges and requirements for the definition of the needs of adhering to the FAIR (findable, accessible, interoperable and reusable) data principles in a large distributed bioinformatics infrastructure. We address the challenge of heterogeneity of wet lab technologies, data, metadata, software, computational workflows and the levels of implementation and monitoring of FAIR principles within the different bioinformatics sub-disciplines joint in de.NBI. On the one hand, this broad service landscape and the excellent network of experts are a strong basis for the development of useful research data management plans. On the other hand, the large number of tools and techniques maintained by distributed teams renders FAIR compliance challenging.

Implementing FAIR data management within the German Network for Bioinformatics Infrastructure (de.NBI) exemplified by selected use cases.

Prostate cancer (PCa) is the most common cancer and the third most frequent cause of male cancer death in Germany. MicroRNAs (miRNA) appear to be involved in the development and progression of PCa. A diagnostic differentiation from benign prostate hyperplasia (BPH) is often only possible through transrectal punch biopsy. This procedure is described as painful and carries risks. It was investigated whether urinary miRNAs can be used as biomarkers to differentiate the prostate diseases above. Therefore urine samples from urological patients with BPH (25) or PCa (28) were analysed using Next-Generation Sequencing to detect the expression profile of total and exosomal miRNA/piRNA. 79 miRNAs and 5 piwi-interacting RNAs (piRNAs) were significantly differentially expressed (adjusted p-value < 0.05 and log2-Fc > 1 or < -1). Of these, 6 miRNAs and 2 piRNAs could be statistically validated (AUC on test cohort > = 0.7). In addition, machine-learning algorithms were used to identify a panel of 22 additional miRNAs, whose interaction makes it possible to differentiate the groups as well. There are promising individual candidates for potential use as biomarkers in prostate cancer. The innovative approach of applying machine learning methods to this kind of data could lead to further small RNAs coming into scientific focus, which have so far been neglected.

Small RNAs as biomarkers to differentiate benign and malign prostate diseases: An alternative for transrectal punch biopsy of the prostate?

Mass spectrometry is frequently used in quantitative proteomics to detect differentially regulated proteins. A very important but unfortunately oftentimes neglected part in detecting differential proteins is the statistical analysis. Data from proteomics experiments are usually high-dimensional and hence require profound statistical methods. It is especially important to already correctly design a proteomic experiment before it is conducted in the laboratory. Only this can ensure that the statistical analysis is capable of detecting truly differential proteins afterward. This chapter thus covers aspects of both statistical planning as well as the actual analysis of quantitative proteomic experiments.

Important Issues in Planning a Proteomics Experiment: Statistical Considerations of Quantitative Proteomic Data.

Therapeutic strategies for patients with locally advanced rectal cancer (LARC) who are achieving a pathological complete response (pCR) after neoadjuvant radio-chemotherapy (neoCRT) are being increasingly investigated. Recent trials challenge the current standard therapy of total mesorectal excision (TME). For some patients, the treatment strategy of "watch-and-wait" seems a preferable procedure. The key factor in determining individual treatment strategies following neoCRT is the precise evaluation of the tumor response. Contrast-enhanced computer tomography (ceCT) has proven its ability to discriminate benign and malign lesions in multiple cancers. In this study, we retrospectively analyzed the ceCT based density of LARC in 30 patients, undergoing neoCRT followed by TME. We compared the tumors´ pre- and post-neoCRT density and correlated the results to the amount of residual vital tumor cells in the resected tissue. Overall, the density decreased after neoCRT, with the highest decrease in patients achieving pCR. Densitometry demonstrated a specificity of 88% and sensitivity of 68% in predicting pCR. Thus, we claim that ceCT based densitometry is a useful tool in identifying patients with LARC who may benefit from a "watch-and-wait" strategy and suggest further prospective studies.

Computertomography-Based Prediction of Complete Response Following Neoadjuvant Chemoradiotherapy of Locally Advanced Rectal Cancer.

Skeletal muscle is a heterogeneous tissue consisting of blood vessels, connective tissue, and muscle fibers. The last are highly adaptive and can change their molecular composition depending on external and internal factors, such as exercise, age, and disease. Thus, examination of the skeletal muscles at the fiber type level is essential to detect potential alterations. Therefore, we established a protocol in which myosin heavy chain isoform immunolabeled muscle fibers were laser microdissected and separately investigated by mass spectrometry to develop advanced proteomic profiles of all murine skeletal muscle fiber types. All data are available via ProteomeXchange with the identifier PXD025359. Our in-depth mass spectrometric analysis revealed unique fiber type protein profiles, confirming fiber type-specific metabolic properties and revealing a more versatile function of type IIx fibers. Furthermore, we found that multiple myopathy-associated proteins were enriched in type I and IIa fibers. To further optimize the assignment of fiber types based on the protein profile, we developed a hypothesis-free machine-learning approach, identified a discriminative peptide panel, and confirmed our panel using a public data set.

Advanced Fiber Type-Specific Protein Profiles Derived from Adult Murine Skeletal Muscle.

(1) Background: Neuroblastomas (NBs) are the most common extracranial solid tumors of children. The amplification of the Myc-N proto-oncogene (MYCN) is a major driver of NB aggressiveness, while high expression of the neurotrophin receptor NTRK1/TrkA is associated with mild disease courses. The molecular effects of NTRK1 signaling in MYCN-amplified NB, however, are still poorly understood and require elucidation. (2) Methods: Inducible NTRK1 expression was realized in four NB cell lines with (IMR5, NGP) or without MYCN amplification (SKNAS, SH-SY5Y). Proteome and phosphoproteome dynamics upon NTRK1 activation by its ligand, NGF, were analyzed in a time-dependent manner in IMR5 cells. Target validation by immunofluorescence staining and automated image processing was performed using the three other NB cell lines. (3) Results: In total, 230 proteins and 134 single phosphorylated class I phosphosites were found to be significantly regulated upon NTRK1 activation. Among known NTRK1 targets, Stathmin and the neurosecretory protein VGF were recovered. Additionally, we observed the upregulation and phosphorylation of Lamin A/C (LMNA) that accumulated inside nuclear foci. (4) Conclusions: We provide a comprehensive picture of NTRK1-induced proteome and phosphoproteome dynamics. The phosphorylation of LMNA within nucleic aggregates was identified as a prominent feature of NTRK1 signaling independent of the MYCN status of NB cells.

Article	Year
Comparison and Evaluation of Clustering Algorithms for Tandem Mass Spectra. Journal of proteome research\| PubMed ID: 28959885	2017
Protein variability in cerebrospinal fluid and its possible implications for neurological protein biomarker research. PloS one\| PubMed ID: 30496192	2018
Integrated Fourier Transform Infrared Imaging and Proteomics for Identification of a Candidate Histochemical Biomarker in Bladder Cancer. The American journal of pathology\| PubMed ID: 30770125	2019
Monocytes-neutrophils-ratio as predictive marker for failure of first induction therapy in AML. Blood cells, molecules & diseases\| PubMed ID: 31029023	2019
Digital droplet PCR-based chimerism analysis for monitoring of hematopoietic engraftment after allogeneic stem cell transplantation. International journal of laboratory hematology\| PubMed ID: 31225701	2019
Effect of therapeutic suggestions during general anaesthesia on postoperative pain and opioid use: multicentre randomised controlled trial. BMJ (Clinical research ed.)\| PubMed ID: 33303476	2020
Implementing FAIR data management within the German Network for Bioinformatics Infrastructure (de.NBI) exemplified by selected use cases. Briefings in bioinformatics\| PubMed ID: 33589928	2021
Small RNAs as biomarkers to differentiate benign and malign prostate diseases: An alternative for transrectal punch biopsy of the prostate? PloS one\| PubMed ID: 33760831	2021
Important Issues in Planning a Proteomics Experiment: Statistical Considerations of Quantitative Proteomic Data. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 33950479	2021
Computertomography-Based Prediction of Complete Response Following Neoadjuvant Chemoradiotherapy of Locally Advanced Rectal Cancer. Frontiers in oncology\| PubMed ID: 34136378	2021
Advanced Fiber Type-Specific Protein Profiles Derived from Adult Murine Skeletal Muscle. Proteomes\| PubMed ID: 34201234	2021
NTRK1/TrkA Signaling in Neuroblastoma Cells Induces Nuclear Reorganization and Intra-Nuclear Aggregation of Lamin A/C. Cancers\| PubMed ID: 34771457	2021

Karin Schork

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