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The program of the Advanced Proteomics Lecture Course compromises Lectures on key topics in Proteomic research, given by experts in the field. The Lectures will consist of a conceptual part, explaining the technology and an applied part, explaining the research.
In the program, 3 afternoons will be reserved for Workshops. Consequently 3 different workshops can be picked from the workshop list of 10 different workshops.
Sample preparation – Thierry Rabilloud
In the lecture, the motto "garbage in, garbage out" was cited to describe how it applies to the utmost to sample preparation to make it a key issue in every proteomic experiment, whatever the proteomic scheme used. In the workshop, this motto is complemented by another well-known one: " the devil hides in the details".
Protein identification and characterisation by database search – Lennart Martens
The identification of fragmentation mass spectra to peptide sequences is a key step in the entire proteomics workflow. As the hinge point between the experimental and the quantitative analysis, the identification process comes in at a crucial juncture in a proteomics experiment. It therefore makes sense to try and do as good a job of it as possible!
In order to help you get the most out of your identifications, this workshop will open up the black box of the identification process using a decidedly hands-on approach; so bring your laptop (Windows, Mac and Linux are all supported)! While we’ll make use of the cross-platform, open source SearchGUI and PeptideShaker tools (which offer built-in access to eight different search engines, one de novosequencing engine, and one tag-based search engine), this workshop is by no means just another tutorial for yet another tool!
Instead, you’ll learn all you ever wanted to know about search engines, search parameters, choosing the right database, scoring systems, and false discovery rate estimation. As a bonus, we’ll also discuss the issues and challenges in complex identification scenarios such as proteogenomics, metaproteomics, and open modification searches. There’s even room to look at some de novosequencing, tag-based searches, and spectral library searching. The skills you’ll acquire are therefore guaranteed to be generally applicable to any other search engine you’ll encounter as well!
You’ll of course also learn about protein inference, the (sometimes tricky!) step of matching the identified peptides back to proteins. We’ll look into the different ways in which this problem can be solved, and how the existence of these various approaches can create issues when comparing data sets. Moreover, you’ll also learn why the protein inference process is very important in light of downstream quantification.
Throughout the workshop, you’ll also learn how to easily and efficiently experiment with your data analysis. This will not only allow you to get a feel for your data, but it will also teach you how to perform and interpret essential quality control checks on your acquired data!
Finally, there’s always room for your own specific questions, issues, or challenges.
Post-translational modifications – Forest White
Cellular regulatory networks are typically regulated, at the protein level, by a combination of altered protein expression and protein post-translational modifications (PTM). Protein expression changes tend to take much longer and often can represent a state change in the cell, while protein PTMs are much more dynamic and reversible, providing rapid response to environmental perturbations. There are well over 100 different protein PTMs that have been identified, with phosphorylation, glycosylation, ubiquitination, and lysine acylation (e.g., methylation, acetylation, etc…) among the most well-characterized PTMs to date. Over the past 15 years the truly proteome-wide nature of protein PTMs has begun to be appreciated, with thousands of sites identified for many of the more common PTMs. Moreover, the response speed of protein PTM signaling networks is now beginning to be revealed, with network-wide changes occurring within seconds of cell stimulation. Quantifying protein PTM networks in different disease states has revealed novel therapeutic targets and combinatorial therapeutic strategies. Despite the promising potential of protein PTM analysis, data acquisition from limited amounts of sample remains challenging and the extraction of biological insight from highly complex data sets can also be difficult. In this workshop we will discuss a range of techniques to enrich, identify, and quantify protein PTMs, by mass spectrometry and other methods. We will highlight the advantages and disadvantages of selected techniques and discuss critical data quality considerations. Data analysis techniques will also be discussed.
Technical (e.g., how do I do this?), experimental design (e.g., will the data from this experiment answer my question?), and philosophical (e.g., why am I even doing this experiment?) questions will be addressed.
Protein Quantitation using Data Independent Acquisition – Christie Hunter/Lukas Reiter
Data independent acquisition (DIA) is increasingly used for global quantitative proteomics study. During this workshop, we will highlight the benefits of using DIA for label free quantification and will explain step by step how to conduct a successful DIA proteomics study. We will cover the following topics:
- Basics of Data Independent Acquisition (DIA) – 30 mins
- Data Processing Strategies – 30 mins
- Targeted Extraction using Spectral Ion Libraries
- Non-targeted extraction approaches
- Hands on data processing session using Spectronaut – 60 mins
We will provide trial licenses of Spectronaut for the students to do hands-on data analysis, from run files, through setting up a library-based extraction, to looking at quantification in the post analysis including statistical testing for differential abundance.
Students should leave the classroom with solid knowledge on the particularities of a DIA analysis and on how to successfully carry out a complete experiment.
Cross-linking mass spectrometry workshop – Fan Liu
Chemical cross-linking combined with mass spectrometry (XL-MS) has emerged as a powerful approach to investigate protein conformation as well as protein-protein interactions. Especially in recent year, this technique has moved rapidly towards the analysis of very large protein assemblies and heterogeneous mixtures of protein complexes. This course is designed for scientist who is interested in using this technique to probe the structure of various proteins/protein complexes and to discover novel protein-protein interactions. The course will cover most aspects of the XL-MS workflow, including sample preparation, cross-link enrichment, MS data acquisition and cross-link data analysis.
Attendees will be provided with practical training on setting up a XL-MS acquisition method on Q-Exactive series and Orbitrap Fusion series, using different acquisition strategies. Furthermore, practical training on XL-MS data analysis using search engines such as pLink, XlinkX and Proteome Discoverer will also be demonstrated. Materials can be download at https://sourceforge.net/projects/xlinkx/prior to the workshop. Please bring your own laptop for the practical session.
Proteomics Informatics and Biostatistics - D. R. Mani
A large compendium of statistics and machine learning methods are available for the analysis of proteomics and clinical proteomics data. Many of these methods are adapted from the realm of genomics, and need to be applied with care, since proteomics data has its own quirks and unique features.
In this workshop, we will explore computational and statistical approaches for analyzing proteomic data, followed by hands-on exercises that apply these methods and algorithms to real-world datasets. The workshop is aimed at helping students develop best practices for analyzing proteomics data for a range of applications. Topics covered will include:
- Sample requirements and experimental design
- Data normalization, missing values and batch correction
- Hypothesis testing, adjustment for multiple-testing and linear models
- Unsupervised and supervised machine learningalgorithms
- Correlation and outlier analysis
- Pathway enrichment analysis
- Cloud computingfor implementing many of these algorithms
NOTE: this workshop requires some pre-installed software (send to you in a separate email)
Manual Interpretation of Peptide Fragment Ion Mass Spectra – Kai Scheffler
The focus of this workshop is to teach the basics skills in mass spectra interpretation every “proteomicist” should hold. Even though protein identification is nowadays routinely performed using computer-aided database searches, the user is often required to verify the quality of identification results. This requires very often quite some experience but also detailed knowledge on peptide fragmentation basics. This knowledge is also essential for de-novo sequencing, a technique that can be assisted with software but is also often verified in a manual fashion.
The Orbitrap is the only original type of mass analyzer to be developed in the past 25 years and has resulted therefore in a great deal of interest. It is a detector implemented in hybrid type and benchtop instruments. During the course of the workshop an introduction into the Orbitrap instrument platform is presented focusing on the different fragmentation techniques, namely CID (collision induced dissociation), HCD (higher energy collision induced dissociation) and ETD (Electron Transfer Dissociation). Further on basic rules in peptide fragmentation will be discussed in detail as well as differences in spectra obtained from CID, HCD and ETD as the most widely used fragmentation types.
This first part focusing on the theory of peptide fragmentation is the essential basis for the second part, the hands-on spectra interpretation (with printed spectra, pen and calculator – no computers!) on selected examples which will be performed to inherit and practice the skills. What seems tough on the first example becomes easier and much clearer with every further example during practicing. This workshop will sharpen your view and understanding of fragment ion spectra regarding look, quality and characteristic details to watch out for and will enable you to judge database search results with much higher confidence
The workshop will consist of three parts:
- Basic introduction into the Orbitrap technology with focus on the different fragmentation techniques CID, HCD and ETD
- Discussion of peptide fragmentation rule
- Hands-on spectra interpretation
All materials needed for the workshop will be provided.
Targeted Protein Quantification using Skyline -Brendan MacLean and Christina Ludwig
This workshop aims at giving you a basic introduction into targeted mass spectrometry and targeted data analysis using the freely available software Skyline. We will discuss the basic principles of the most frequently used targeted techniques, which are Selected Reaction Monitoring (SRM or MRM) and Parallel Reaction Monitoring (PRM). Together, we will setup and analyze a small targeted experiment using the Skyline software. For this practical part participants are encouraged to bring their own laptops and to already install the Skyline software, which is freely downloadable under
While the focus of this workshop will be on analyzing targeted proteomics data with Skyline, also alternative data acquisition methods, such as DDA or DIA, can be discussed and demonstrated, if this is of specific interest to the workshop participants.
NOTE: this workshop requires some pre-installed software (see below)
- Workshop participants are encouraged to bring their own laptop on which they ideally have already installed the latest Skyline version available at https://skyline.ms/Skyline.url.
ATTENTION: Skyline runs on Windows! However, is possible to use a Mac laptop, but this takes considerably more preparation since you will need to either set the Mac up for dual boot to Windows or a Windows VM (VMWare has been recommended for this in the past)
- All materials (other than the laptop) will be provided during the workshop.
ATTENTION: This will include a USB flash drive, so please make sure that you have the rights on your laptop to copy and paste data files from a flash drive.