From lots of gibberish to useful data…
This post is destined to become the introduction of a new series of glorious blog entries on the subject of LC-MS/MS data analysis.
In this series we would like to talk about proteomics data analysis and the different steps it involves. The series will focus on the data type most non-specialist users are likely to have, namely Bottom-Up proteomics data acquired in Data Dependent Acquisition mode. The data may correspond to unlabelled samples, or to samples labelled with SILAC or isobaric labels (TMT or iTRAQ). As this series progresses, we may also touch on other types of proteomics data. We will focus on the theoretical steps of the analysis, but if we get around to it we may also add a tutorial for performing some analysis steps yourself.
Imagine you have recently sent samples to us or another proteomics service or facility, and have just received the results. These may include a report on the analysis with results, tables of proteins and peptides as well as files containing all of the raw acquired spectra.
But… hey, you are a true scientist and you don’t like blackboxes : you must know how it works!
The aim of this series is to help you understand the different steps of the process that we need to get through to convert proteomics spectra into useful expression data. As this series progresses, we will go through:
Data Dependent Acquisition (DDA) is the standard method for running LC-MS/MS experiments. Here, we present a quick overview of the DDA method, as well as of the less common Data Independent methods (DIA). In LC-MS/MS, purified peptides are separated on an online chromatography gradient (usually low pH Reverse Phase) over 1 to 4h and progressively […]
Ideally, each MS2 spectrum would contain enough information to go back unambiguously to the sequence of its parent precursor. Since this is actually rarely the case, a strategy called Database Searching is usually used instead. MS2 fragmentation spectra are used to identify the sequence behind their MS1 parent peak. However, MS2 spectra rarely allow unambiguous […]
New project to explore overlooked area of Alzheimer’s Disease research Alzheimer’s disease (AD) affects over 35 million people world-wide at present, these numbers are expected to grow substantially over the next few decades and there are currently no medicines which slow the progress of the disease. To boost the development of new medicines for AD, […]
Thermo Fisher releases TMT 11-plex. Thermo Fisher recently released an additional channel for TMT, which means you can now combine up to 11 different samples as one for better sample comparability and more complete data! It is now possible to use “isotopologue” encoding to split the 131 (last) channel into 131-N and 131-C to get […]
Mass spectrometry-based proteomics is an amazing and still fast evolving field. The way clever people constantly come up with new ways to tweak experiment design to tackle wholly different biological questions never ceases to amaze me. When I started in MS, I had mostly heard of people using MALDI-TOF instruments. Orbitraps were just emerging as […]
Last week, my colleague Francesco Rao (DC Biosciences’ CSO) and I travelled to Bonn to participate in a progress meeting for ADAPTED (Alzheimer’s Disease Apolipoprotein Pathology for Treatment Elucidation and Development). Bonn, the birthplace of Beethoven (though everyone’s favourite tortured pre-romantic composer spent most of his years in Vienna), is a relatively small city […]
From lots of gibberish to useful data… This post is destined to become the introduction of a new series of glorious blog entries on the subject of LC-MS/MS data analysis. In this series we would like to talk about proteomics data analysis and the different steps it involves. The series will focus on the […]
Today we will be speaking of proteomics data files and their contents. We will also talk a bit about proteomics file formats, and why they are a mess. In a typical case, you would have been running peptide samples on a mass-spectrometer using a gradient to separate peptides by some physicochemical property so they don’t […]
Interpreting Individual Peaks – part 1 If you have ever looked at a mass-spectrometer output file, you may have wondered how it is possible to make sense of all of these peaks. I mean, there really is a lot of them! Here, we will talk a bit about the basic rules which govern the behaviour […]
Hi everyone, and welcome to a glorious New Year! 2017 was fun, wasn’t it? I for one so much look forward to 2018 being slightly more normal. Oh what the hell, who am I kidding, I’ll get some popcorn already. Today, we will be talking about MS2 spectra, that is, the spectra resulting from the […]
In previous instalments, we looked at what happens to the poor peptides we send to their doom down the rabbit hole and into the strange wonderland that is the inside of a mass spectrometer. Now, it’s all fine and good to fragment peptides into tiny little pieces, I for one am all for it. I […]
This week Francesco Rao attended a progress meeting for the ADAPTED project. For this meet up the city of choice was Edinburgh, some might argue the most beautiful city in the world! After a successful launch year, this was the first consortium meeting in 2018. With someone in the world developing Dementia every 3 seconds, the […]
So, you’ve got this beautiful proteomics experiment done. You just got your data (yay!), let’s say ratios of treated vs control, 3 replicates, simple experiment. But that’s thousands of protein groups, most of which are probably not interesting at all. We want to find out which proteins are regulated. How do we do it? […]
Click chemistry reactions are reactions between an alkyne-tagged molecule and an azide-tagged molecule which use copper as a catalyst. This was introduced by K. B. Sharpless in 2001 for which he was awarded half of the 2001 Nobel Prize in Chemistry. Sharpless developed these reactions in The Scripps Institute, California and has continued developing more […]
There was an interesting paper recently: A new type of Isobaric Labelling reagents? Do we really need more? Isn’t this what TMT and iTRAQ already do? Why is this important? Isobaric Labelling In Isobaric Labelling, each sample is modified with different versions of a chemical label which are near identical but have […]
Hi, I’m Jan and I’m new around here. In fact, I’m new science altogether, but I am definitely learning new things every day and enjoying being a part of such a dynamic community. I wanted to learn a bit more about the history of DC Biosciences, how it became so pioneering in the field of […]
The COVID-19 pandemic is a global, public health crisis with far-reaching and long-term repercussions on the financial, physical, and psychological well-being of the population. As a consequence of the highly contagious nature of the disease, the virus has spread rapidly across the world. The disease is associated with severe pneumonia that often leads to acute […]
Proteomics is the analysis of the entire protein complement of a cell, tissue, organism, or species. The application of technologies to identify and quantify proteins is crucial in early disease diagnosis, prognosis, and drug development. Proteomics as a discipline has its roots in the late 1800s with the emergence of rudimentary mass spectrometry. The following […]
Our commitment to pursue the most pioneering, leading-edge proteomic technologies has driven us to add de novo Protein and Antibody Sequencing to our service provision. “This year we will focus on ensuring a seamless results-driven service and apply our extensive knowledge and experience across a broad range of pioneering proteomic disciplines to provide our customers with […]
Latest News from DC Biosciences: The team at DC Biosciences is excited about bringing new ideas and an improved focus as the world moves forward to an easier and more productive time. Our aim is to provide a more dynamic and diverse proteomic service enabling us to provide the best match for your unique research […]
The ADAPTED (Alzheimer’s Disease Apolipoprotein Pathology for Treatment Elucidation and Development) project was a major new initiative to investigate an area of Alzheimer’s Disease (AD) research that has previously received little attention. The project aimed to improve understanding of the function of the APOE gene. APOE is well known as a risk factor for developing […]
ADAPTED Consortium Identifying new medicines to treat Alzheimer’s Disease through greater understanding of the APOE gene. Alzheimer’s disease (AD) is the most common form of dementia, responsible for at least 60% of cases. Today, there is a worldwide effort underway to find better ways to treat the disease, delay its onset and prevent it from […]
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