Are you missing out on how Progenesis QI can help your research?

ASMS 2019 has come and gone but the research continues. Nonlinear and Waters were lucky to have speakers at ASMS 2019 who shared their research with us and explained how the Progenesis QI software was an integral part of their work.

If you didn’t get to attend the talk by David W. Gaul, PhD., from the Georgia Institute of Technology or would like to hear the presentation again then we have the video for you. The title of his talk was “Preoperative Metabolomic Signature of Prostate Cancer Recurrence” To get an idea of what the presentation entails, you can read the abstract below, but I highly recommend you watch the video so you can see how important the work David and his group are doing with regards to giving people more information on how they can change their treatment decisions moving forward from a simple blood test.

Abstract

Up to 50% of prostate cancer surgical patients will suffer from biochemical recurrence manifested by detectable serum prostate specific antigen levels even after prostate removal. Currently available pre-operative information has not yielded adequate prognosis to guide a patient’s treatment decision.  More clinical data is needed to improve prognosis. New biomarkers can arise from metabolome analysis of patient serum to aid physicians and patients in developing a treatment plan. We applied a multiplatform (NMR + LC−MS) metabolomics approach to the study of preoperative metabolic alterations associated with prostate cancer recurrence.

If you would like to trial the Progenesis QI software in your lab to see how it can impact your research, then please get in touch.

Why are reviews important?

The internet is a huge resource at our fingertips that enables us to find lots of information about any product that’s of interest to us, but how can we be confident the information is accurate, unbiased and a true representation of the product? 

That’s where reviews come in. If a real live person has used a product and given a review, then you can be more confident about that product and its capabilities and the choice you are making.

Here at the Progenesis QI team, we want you to be happy with your mass spec software so we’ve been working with SelectScience for a while now to generate informative reviews of Progenesis QI and Progenesis QI for proteomics software that will be a valuable resource for our potential users.

Who are SelectScience?

Image of the SelectScience logo celebrating their 20 year anniversary
SelectScience an independent product review website

I’d be surprised if you haven’t heard of SelectScience already but if you haven’t, they are a trusted, independent website for laboratory scientists to find impartial reviews written by their peers, as well as application notes and videos. They work with scientists to inform them about the best products and applications. Google recognizes them as a trusted review site for independent product reviews and they have won the Queen’s Award for International Excellence.

two images. One showing the logo for the Queens Award for Enterprise and the other shows the five star trusted review image.

As a Progenesis QI or Progenesis QI for proteomics user, you’ll be receiving an email shortly with a link to enter a prize drawing for the chance to win an *iPad® or a $400 *Amazon® gift card in return for simply writing a review and sharing information about Progenesis QI or Progenesis QI for proteomics software with your peers.  If you don’t receive the email, then let me know and I can send you the link. You will have to act quickly as the option to enter the draw will only be available for a limited time.

If you have used the software in the past then you also have the option to write a review here or you can access the review form direct from our website.

You can see below some of the reviews we currently have for both the Progenesis QI and the Progenesis QI for proteomics software. As more and more people use it, we hope many of them will share their experiences.

Image of the review from SelectScience for the Progenesis QI for proteomics software.

Review for the Progenesis QI for proteomics software
Image of a review from SelectScience for the Progenesis QI software

Review for the Progenesis QI for proteomics software

If you haven’t tried the software yet and would like to talk further, don’t hesitate to get in touch.

*iPad® and Amazon® are not affiliated with this drawing and are independently owned and operated.


Identification scoring in Progenesis QI

With the amount of information available today, important and helpful information can easily get lost and overlooked. I’d like to take this opportunity to repost this blog post about identification scoring in Progenesis QI of as many of our customers find this very useful in their research and still refer to it today.

One of the advantages of using Progenesis QI is its ability to combine results from multiple search methods and databases. Progenesis QI uses a common scale to score results from all the databases and search methods it supports, so you can compare search results obtained from different search methods. This post explains the scoring method we use in Progenesis QI, and how you can improve your search scores by searching additional dimensions of your data.

Progenesis QI search methods

At the time of writing, Progenesis QI supports these search methods and databases:

Progenesis MetaScope

Searches SDF and MSP files from any source. Supports retention time, CCS, theoretical fragmentation and spectral libraries.

METLIN™  MS/MS Library (requires purchase)

The Waters® METLIN™ MS/MS Library for Progenesis QI contains a local copy of the METLIN database and allows you to search this copy rapidly.

LipidBlast

Searches the LipidBlast MS/MS database provided by Metabolomics Fiehn Lab.

Elemental composition

Produces putative formulae for compounds based on mass, isotope profile, and the Seven Golden Rules.

ChemSpider

Searches the ChemSpider structure database. Supports theoretical fragmentation, isotope similarity filtering, and elemental composition filtering.

NIST MS/MS Library (requires purchase)

Searches the NIST MS/MS library for spectral matches.

You can find out more about each of these search methods in the search methods and databases FAQ. This blog post, however, will focus on how we calculate scores so that identifications from different search methods can be compared.

The Progenesis scoring method

For any given search, there are a possible five properties that can contribute to the overall score:

  1. Mass error
  2. Isotope distribution similarity
  3. Retention time error
  4. CCS error
  5. Fragmentation score

Each of these individual scores is on a scale from 0-100. If your search criteria do not include a given piece of data, the score for that piece of data is 0. The overall score is the mean of these 5 scores.

Note that the more search criteria you use, the higher the maximum possible score becomes, as described in the following example.

Example

Suppose we have searched ChemSpider using theoretical fragmentation. For a given compound we find Identification A, with these scores:

Note that the scores for retention time and CCS errors are 0, because ChemSpider does not support searching those properties.

If we then perform a MetaScope search, this time including a CCS constraint, we might obtain the following scores for Identification B:

We have identical scores for the mass error, isotope distribution, and fragmentation. However, we also have an extra piece of information in the CCS score. This provides additional evidence for Identification B, so it is given a higher score than Identification A.

Note that in the ChemSpider case, if an identification scores 100 on all 3 items, it obtains a score of 60. In the MetaScope case, if an identification scores 100 on all items, it obtains a score of 80. So, for each additional piece of data we include in our search, the maximum score increases by 20.

The component scores

Here we’ll briefly describe how the five component scores that make the final score are calculated.

Mass error, retention time error, and CCS error

These are all functions of the magnitude of the relative error, Δ:

The score profile for mass error, retention time error and CCS error.
Figure 1: The score profile for mass error, retention time error and CCS error.

For the mass error, Δ is the ppm mass error and N = 4000. For the retention time and CCS errors, Δ is the percentage error, and N = 20.

Isotope distribution similarity score

This compares the intensities of each isotope between observed and theoretical distributions. A total intensity difference of 0 gives a score of 100, which falls linearly to 0 when the total intensity difference is equal to the maximum isotope intensity.

Fragmentation score

The fragmentation score is more complicated and depends on the fragmentation method used. The FAQs describe how scoring works for theoretical fragmentation and database fragmentation.

Improving identification scores

The best way to improve the scores of your identifications and your confidence in them is to use more search constraints.

37.9/100

In general, most searches will be able to produce a mass error score and an isotope similarity score. With just these two pieces of information, the maximum score for any identification is only 40/100. In this example we’ve identified Warfarin using only mass error and isotope similarity.

55.4/100

By including fragmentation data in your search criteria (either theoretical fragmentation or a fragmentation database), this increases the possible score for identifications to 60/100. Here we’ve added theoretical fragmentation to our search parameters.

70.8/80

Finally, if you use an appropriate data source (e.g. an SDF and additional properties file) you can add search constraints for retention time and CCS, giving a maximum score of 100/100. Here we don’t have CCS information but have added retention time to our search parameters for a maximum of 80/100.

Future improvements

Currently Progenesis gives equal weight to the five component scores – mass error, isotope similarity, fragmentation score, retention time error, and CCS error. In some cases, this might not be ideal, so if you have any suggestions for different weightings we’d love to hear from you in the comments section below.

As always, if you have any further questions, check our FAQ or get in touch.

How do you know your raw materials are as they should be?

Agnès Corbin of Nonlinear Dynamics gives us an overview of what’s needed to maintain high manufacturing standards.

Agnes Corbin

We all know it, reproducibility is one of the key parameters to master for maintaining a product’s quality. As a customer, we all like our favorite products of a consistent high quality; as a manufacturer, we want to preserve our quality and customers’ satisfaction

That starts with the supply chain of the raw materials and ingredients used to manufacture a finished product.

A non-conformity, be it a cross-contamination, adulteration or degradation, can have huge economic, clinical and sanitary consequences, especially with high cost raw materials.

With this in mind, you might be interested in the below application note, produced in collaboration with Robertet Group, the world leader in sustainable natural raw materials for fragrance and flavor.

Click on the image to download the Vetiver essential oils application note
Vetiver essential oils application note

It describes how Progenesis QI was used, to spot an ‘out-of-the-blue’ potential non-conformity in Vetiver essential oil, using an Untargeted Metabolomics Profiling approach with LC-HRMS and a variety of Ionization techniques.

Progenesis QI helped to detect and identify adulteration with Castor Oil, a non-volatile compound, in a new batch of Vetiver Essential Oil. It would have been missed with the use of classical and common GC-MS techniques applied on volatile compounds.

The combination of LC separation equipment UPC² and UPLC (with different ionization sources ESI, APCI, ASAP) were used to get a better understanding of the product’s composition. Suppliers of natural raw materials must increase their phytochemistry knowledge of their products, as per the recent change in the REACH regulation based on the Natural Complexes Substances (NCS).

The easy-to-set-up LC-MS techniques for non-volatile compounds, can be considered as complementary to GC-MS for volatile compounds QC.

Robertet could have missed out if they hadn’t used the Progenesis QI software.

Are you missing out by not using it?

Please contact us for an evaluation today and we can help you with your research.

The importance of the surfaceome and its interactors

We love to hear how our customers are using Progenesis QI and Progenesis QI for proteomics. It’s great to learn what they are researching and how Progenesis can help them. It’s also nice to find out more about the people behind the research. Our latest blog post features Dr Maria Pavlou and her interesting work with Dualsystems Biotech AG. First, here’s some background about Maria:

Dr Maria Pavlou

Maria Pavlou received her PhD in translational proteomics from the Department of Laboratory Medicine and Pathobiology at University of Toronto, Canada. Upon PhD completion, Maria moved to Switzerland to pursue a post-doctoral fellowship in the Institute of Molecular Systems Biology at the Swiss Federal Institute of Technology (ETH) in Zurich focusing on host-pathogen interactions. In 2017, she joined Dualsystems Biotech AG as a senior scientist and a year later, she was promoted to Chief Scientific Officer leading the research team to develop further the Ligand-based Receptor Capture (LRC) methodology and establish new services.

Now, onto the research:

The importance of the surfaceome and its interactors

If the plasma membrane is considered the gateway through which cells communicate and interact with their environment, then proteins associated with the surface – referred as the surfaceome – can be seen as the gatekeepers. The surfaceome largely dictates the shape, polarity, differentiation and motility of cells. It also mediates cellular behaviors such as cell-cell communication, self and non-self recognition, and cell signalling. Given the crucial role of surface-associated proteins in every aspect of cellular life, it is not surprising that they are the molecular targets for roughly 70% of FDA approved drugs [1].

The original concept, depicting the plasma membrane as a homogeneous fluid bilayer with freely diffusing proteins, has been evolved to another depicting a highly organized and crowded mosaic of interacting lipids and glycoproteins. This higher organization modulates the biological processes occurring on the cell surface, exemplified by receptors being active only when they form dimers, hetero-dimers or higher order oligomers [2]. Interactions of proteins in the cell membrane of the same cell (cis), and interactions of proteins of neighboring cells, the extracellular matrix and circulating ligands (trans) are collectively referred to as extracellular protein-protein interactions (ePPIs).

Elucidating ePPIs in a systemic fashion is pivotal to gain a better understanding of the surfaceome function. More specifically, identifying the targets of key ligands on the cell surface provides valuable mechanistic information about signal transduction, drug action or off-target effects. For instance, pathogen or growth factor interactions are important for developing novel therapies. Additionally, numerous ligands exist – both biologics and small molecules – involved in biological functions mediated at the cell surface through still unknown protein targets.

Towards target identification, an advanced cell-based chemo-proteomic approach has been developed namely ligand-based receptor capture (LRC) [3-4]. In this approach, the endogenous receptor repertoire of a given cell serves as an existing bait library that can be probed for ligand interaction. The key component of the LRC methodology is a trifunctional compound (TriCEPS or its latest development named HATRIC [3-4]) that utilizes the extensive glycosylation displayed by the majority of cell surface proteins to capture receptor interactions on living cells. Experimentally, the first arm of TriCEPS is conjugated with the primary amines of a ligand and the conjugates are added on living cells (mildly oxidized). There the ligand binds to its target(s) and the second arm of TriCEPS is covalently crosslinked to the glycans of the binding partner. The third arm facilitates target purification for mass spectrometric analysis.

In a typical LRC-TriCEPS experiment, at least two treatment arms are performed in parallel: one with the ligand of interest and a second with a control ligand (that is, a ligand with a known target). Upon identification, the relative abundance of cell surface proteins in the ligand samples is compared to those in the control samples using MS1-based label-free quantification. Randomly identified cell surface proteins are expected to have equal abundance in both samples, whereas the corresponding receptors are found enriched in the ligand sample.

Progenesis QIP gives the user full control but does not require advanced computational skills

Progenesis QI for proteomics (QIP) has been the workhorse when it comes to data analysis. Performing MS1-based label-free quantitation in Progenesis is extremely straightforward through an intuitive user-friendly interface.

The alignment of features is performed by sophisticated algorithms but at the same time the software provides the user with visual inspection of the whole procedure. This is extremely useful as the user has full control of the data and a better understanding on how the samples are processed. It can also reveal technical issues related to the liquid chromatography separation prior to mass spectrometric analysis or sample quality. Notably, proper feature alignment is pivotal for robust quantitation. Moreover, the feature-picking algorithm (peak picking) has been developed to minimize missing values therefore the requirement for imputation; another asset for robust quantitation.

Through the various filtering options, the user can eliminate features that are not of interest (such as polymers or contaminants) focusing on what really matters. The QC metrics tab provides a qualitative overview of the experiment giving the opportunity (once more) to assess whether the LC and MS parameters used were optimal.

Upon protein inference and calculation of relative abundances, the user can easily review protein characteristics (such as number of peptides, peptide sequence and modifications, expression profiles, see figure 1) and confirm results or flag outliers. Once more, this option gives the user full control over the data and eliminates, to a great extent, experimental artefacts.

LRC-TriCEPS analysis to identify the receptors of Insulin and Transferrin on HEK293 cells; a screenshot of Progenesis QIP.

Two LRC experiments were performed using insulin and transferrin as ligands of interest on HEK293 cells with receptor capture at two different pH (6.5 and 7.4).

(A) Following the intuitive and straightforward progenesis pipeline, the identification and quantitation (MS-1 based) was completed within 6 hours.

(B) A total of approximately 300 surface proteins were identified and quantified across all samples. Transferrin receptor (TFR1), the known target of Transferrin, was also identified with roughly 40 unique peptides.

(C) Using the protein filter the relative abundance of TFR1 across the four conditions was visualized; TFR1 is clearly enriched in the Transferrin samples.

(D) For more detailed information the user can check the quantitation of every peptide identified and spot any irregularities.

(E) The user can use the statistics run by Progenesis or export the data for post-hoc analysis.

As statistical testing is incorporated in the software, the final outcome of an analysis provides immediate information regarding proteins being significantly regulated. However, there is still the option to export all necessary information in order to perform post-hoc statistical analysis using different tools. This increases greatly the flexibility of the user.

Finally, Progenesis QIP is readily scalable when it comes to number of samples and performs analysis in a time-efficient manner, allowing for complete label-free quantitation in the course of a working day. This is extremely important given that data analysis is usually the beginning of a series of experiments aiming to verify and interpret the identified quantitative differences. It provides a variety of different plots and graphs that can be readily used for publications or reports.

In summary, the analysis of LRC-TriCEPS data with Progenesis QIP offers unique advantages. The software is user-friendly and intuitive therefore can be used by researchers with experience in data analysis but also by users that are just starting or do not perform data analysis daily. Progenesis QIP provides the user with full control over data analysis which is very important to spot and resolve experimental artefacts and to understand how the final outcome is reached. At the same time, the sophisticated algorithms provide high quality label-free quantitation and robust results. Finally, the nice visualization aspects can generate high quality graphs that can be used to communicate the results of each study.

1. Uhlén M, et al. Tissue-based map of the human proteome. Science (80- ). 2015.

2. Milligan G, G protein-coupled receptor dimerisation: molecular basis and relevance to function, Biochim Biophys Acta. 1768(4):825-35, 2007

3. Frei AP, Moest H, Novy K, Wollscheid B. Ligand-based receptor identification on living cells and tissues using TRICEPS. Nat Protoc. 2013;8:1321–36.

4. Sobotzki N, et al. HATRIC-based identification of receptors for orphan ligands. Nat Commun. 2018;9:1–16.

Thank you to Maria for a very interesting blogpost. Finally, please get in touch

• If you are a user with an interesting research project using Progenesis. We are keen to share user stories via our blog.

• If you would like to try Progenesis on your own data

Thank you

Acknowledgement: Maria Pavlou, PhD, Paul Helbling, PhD

A Progenesis QI workflow in Exposomics

Following on from the previous post about our 3 Progenesis QI lunchtime presentations at IMSC 2018, we are proud to present to you the talk given by Emilien Jamin from the Toxalim, Research Centre in Food Toxicology, Toulouse University, available to view here.

Emilien’s work in contaminant discovery and analysis shows how Progenesis QI can be used very effectively for untargeted analysis in the Exposomics field.   This workflow goes beyond suspect screening which requires prior knowledge.  Emilien uses several examples of how Progenesis QI was used to discriminate between different populations and finally touches on a proof of concept on lipids peroxidation.

You can view the talk for yourself and you can read an overview of his presentation below.

Metabolomic profiling of reactive metabolites in toxicology by MSE and Progenesis QI

Metabolomic profiling of reactive metabolites in toxicology by MSE and Progenesis

Emilien Jamin, Robin Costantino, Jean-François Martin, Françoise Guéraud, Laurent Debrauwer

Toxalim (Research Centre in Food Toxicology) Toulouse university, INRA, ENVT, INP-Purpan, UPS, F-31027 Toulouse, France.

Axiom Platform, MetaToul-MetaboHUB, National Infrastructure for Metabolomics and Fluxomics, F-31027 Toulouse, France

In food safety, current exposure assessment approaches are based on food consumption data crossed with food contamination data or biomonitoring data. This allows evaluating exposure only in a targeted way on a few families of compounds. Based on our previous results in exposomics [1], food or environmental toxicology should focus on the exposure to a mixture of compounds (contaminant cocktails), mostly at low doses, and in an untargeted way to detect/identify unknown compounds. And among these numerous known and unknown metabolites, it seems a priority to focus on potentially toxic compounds.

In this context; we developed an untargeted method using high resolution mass spectrometry coupled to liquid chromatography to specifically profile electrophilic metabolites, in parallel with a classic untargeted metabolomic study. This allows the study of the exposure of potentially toxic compounds on one hand, and the study of the effects of this exposure on the endogenous metabolites on the other hand. More precisely, we used the MSE mode of a Synapt G2-Si mass spectrometer to detect all the metabolites displaying a neutral loss specific of metabolites conjugated with mercapturic acid. Data from MSE and from untargeted HRMS analyses were processed with Progenesis QI, to highlight discriminant reactive metabolites, as well as endogenous metabolites.

As a proof of concept, this approach has been applied to the study of different groups of rats fed diets containing various oils. According to our previous results on lipid peroxidation [2] these diets led to the production of different aldehydes conjugated to mercapturic acid. The most well known is DHN-MA which corresponds to the mercapturate conjugate of 4-hydroxynonenal (4-HNE), which is commonly used as a biomarker of lipid peroxidation [2]. Using our methodology, we were able to detect without a priori, dozens of mercapturate conjugates, including DHN-MA and other known conjugated aldehydes. Furthermore, our approach also allowed the detection of conjugates of unexpected aldehydes, and of other chemical classes, for which putative identifications have been proposed based on complementary structural analyses. Interestingly, multivariate statistical analyses of the HRMS signals carried out on the mercapturate conjugates yield a better characterization of the studied animal groups compared to results obtained from a classic untargeted metabolomic approach.

[1] Jamin E.L. et al. Anal Bioanal Chem (2014) 406:1149–1161

[2] Guéraud F. Free Radic Biol Med (2017) 111:196-208

Progenesis QI is a powerful tool for contaminant analysis and has been used in the food, cosmetics, natural products, chemical materials, sports doping, biopharma, metabolomics and proteomics fields.

Why not download the software and see how it can help you in your research? Progenesis QI for Progenesis QIP for proteomics

Can Progenesis QI impact your research project?

At IMSC 2018, we were lucky to have not one, not two, but three researchers give their presentations at our Progenesis QI lunchtime seminar.

Progenesis–Three personal accounts showing the power of Progenesis QI

  • Untargeted metabolomics using Progenesis QI for small molecules: Developing ion-chromatography-mass spectrometry for the investigation of cancer metabolism – James S.O. McCullagh, University of Oxford, UK
  • Metabolomic profiling of reactive metabolites in toxicology by MSE and Progenesis – Emilien Jamin, Toxalim (Research Centre in Food Toxicology) Toulouse university, INRA, France
  • Novel strategies for discovery of cardiovascular biomarkers in human plasma – Donald JL Jones, Leicester Cancer Research Centre, RKCSB, University of Leicester, UK

These were recorded so we’d like to draw your attention to the interesting and varied presentations over the next few blog posts.

As one of the presenters is awaiting publication, we will present these in reverse order, starting with a lively 23-minute presentation by Prof Don Jones of the University of Leicester.

Below is a short written summary of Don’s talk.  Even better, watch it for yourself and learn which features of Progenesis QI for proteomics Don found so helpful in this ambitious project.  It really is 23 minutes well spent!

Screenshot of the title page for the talk

Novel Strategies for Discovery of Cardiovascular Biomarkers in Human Plasma

 

Donald JL Jones1,2, Sanjay Bhandari2, Paulene Quinn2, Jatinderpal Sandhu2 and Leong L Ng2

1Leicester Cancer Research Centre, RKCSB, University of Leicester, Leicester, LE2 7LX, United Kingdom

2Department of Cardiovascular Sciences and NIHR Leicester Cardiovascular Biomedical Research Unit, Glenfield Hospital, Leicester, LE3 9QP, United Kingdom

Background: The search for blood-based biomarkers is particularly compelling in the cardiovascular clinical arena. Whilst understanding the genetic basis of cardiovascular disease will provide a clear indication of risk, phenotypic markers represent the pathological changes that occur during disease processes. Methods for investigating the plasma proteome have ostensibly relied on complex pre-analytical protocols that are expensive and limit throughput.

Methods: 100 Coronary heart disease Patients with 20 healthy control were analyzed on the SYNAPT G2-Si, using label-free data-independent acquisition LC-MS with ion mobility optimized (HDMSE). Samples were treated with Calcium Silicate matrix (CSM). Raw data was then analyzed using Progenesis QI for Proteomics. Models of panels of markers were developed using SPSS and RapidMiner.

Results: From 50 µL of plasma, in excess of 1800 proteins are realized that can be reliably observed between samples. Of these, >1100 are quantified. The data shows high reproducibility with known differences predictably demonstrated. New markers are revealed which can be strongly aligned with potential novel mechanisms of coronary artery disease (CAD). The method is shown to be highly reproducible.

Conclusion: We demonstrate that CSM provides sufficient coverage to enable single shot analysis of plasma, historically, a very challenging proteomic sample to analyze, and can provide potential markers for CAD which could feasibly be extended to several classes of disease. This provides a method that can run alongside other omic technologies to profile large-scale numbers of patients individually and thus usher in a new era of precision medicine. Importantly, there are advantageous savings to be made in terms of cost and throughput, which mean that for the first time large scale cardiovascular cohorts, conducted in a realistic timeframe, can be analyzed using proteomics

If you would like to try the Progenesis QI software on your own data then please don’t hesitate to get in touch.

Acknowledgments

Professor Donald JL Jones

Can Progenesis adapt to your application?

Adaptability is a critical factor in research success.  How adaptable is the Progenesis QI and Progenesis QI for proteomics software? The answer is very adaptable. You’ll be surprised to see what different market segments and applications the software has been used for.

Food and environment

In this market sector, the software has been used for food authenticity which is now a major concern across the globe.  This blogpost “Don’t get stung by your Manuka Honey!” Highlights the use of the Progenesis QI software in laboratories that test food to examine the differences between genuine and fraudulent products. For further reading, “Bringing the analysis to the sample: Progenesis QI helps beat food fraud” discusses Professor Chris Elliott’s webinar where he talks about many instances of food fraud including milk, horse meat, oregano, and cumin fraud. Chris speaks about how he works with Progenesis QI to analyze data produced at the Institute for Global Food Security.

Review of standardized abundance profiles and assignment of identity for three markers of Manuka honey as displayed in Progenesis QI software.

Figure 1. Review of standardized abundance profiles and assignment of identity for three markers of Manuka honey as displayed in Progenesis QI software.

Natural products

As with the Food and Environment sector, adulteration is also being seen in the natural products market sector. This blogpost aptly named “Just because it’s natural doesn’t mean it’s safe” describes the webinar on Authenticated Herbal Supplements with a metabolomics approach.  This looks at correctly identifying the marker compounds of each species of Hoodia, Terminalia, and Chamomile to ensure they are the correct substance. The work was carried out with collaboration from Waters and the University of Mississippi. For further reading you can read the relevant paper: Metabolic Profiling of Hoodia, Chamomile, Terminalia Species and Evaluation of Commercial Preparations Using Ultrahigh-Performance Liquid Chromatography Quadrupole-Time-of-Flight Mass Spectrometry.

Screenshot of the Authenticated Herbal Supplements with a metabolomics approach webinar.

Figure 2: Screenshot of the Authenticated Herbal Supplements with a metabolomics approach webinar.

Chemical and materials science

What value can Progenesis QI provide in the world of co-polymer characterization? Polymeric materials are being used more and more in everyday life. They are being used as structural materials for cars and airplanes, fabrics for clothing, packaging materials for food and medicine, to name but a few. These new materials must be properly characterized in order to manufacture the polymers’ reproducibly and thus achieve the required characteristics.

This blogpost explores using Pyrolysis-Gas Chromatography/Mass Spectrometry (Py-GC/MS) coupled with Progenesis QI’s multivariate analysis of the data, to provide novel insight into the polymer structure.

Score plot showing two co-polymer types that are clearly discriminated.

Figure 3. Following replicate analysis of the two co-polymer samples the data was aligned and peak picked using the workflow presented by Progenesis QI. The resulting data was analyzed using an OPLS-DA model to compare the samples. The scores plot resulting from that analysis is shown here where it can be seen that the two co-polymer types are clearly discriminated.

Biopharma

An exciting application is the use of Progenesis QI for proteomics for Host Cell Protein (HCP) analysis. The blogpost, “Progenesis QI for proteomics speeds up biopharmaceutical purification!” , explains how using the software greatly simplifies the user interaction with HCP datasets. Extracting mass chromatograms and calculating peak areas for a multitude of peptide precursors (like the 113 peptides from the MIX-4 spectral library) can be a tedious process. The data from each individual sample replicates need to be compared in order to obtain the peptide level HCP trends. The HCP peptide level results need to be translated into HCP protein levels. All these steps are automated and they are performed rapidly in the Progenesis QI for proteomics software without significant user intervention. This saves a significant amount of time spent on data analysis, producing rapid results.

Four HCPs that were identified by Progenesis QIP in the highly-purified NIST mAb.

Figure 4. Four HCPs (highlighted by red arrows) were identified by Progenesis QIP in the highly-purified NIST mAb.

Proteomics

Dr. Maartens Dhaenens shares his expertise in using the software when he presented his webinar “Proteomics: A peptides Journey to Emergence”. This blogpost of the same name explains his thoughts about understanding how all the different levels of complexity in proteomics data can be unraveled by the different functionalities of Progenesis QI for proteomics. He extols how Progenesis QI for proteomics is a powerful tool for resolving the many challenges in automated data analysis and protein inference.

Screenshot of the title for the Webinar

Figure 5. Screenshot of the title for the Webinar

Metabolomics

We have many happy customers, including Dr. Daniel Carrizo, who uses the Progenesis QI software for his metabolomics research. Daniel uses the Progenesis QI software to assess exposure to persistent organic pollutants (POPs). Daniel comments “The data generated is too complex to analyze without specific software like Progenesis. When you have 3000 or 4000 ions of interest and 300 samples, it is impossible to manage this amount of data with normal software. I have found Progenesis QI is robust and easy to use and the technical support is excellent.” He goes on to say “The most important aspect is the power of the analysis and robustness of the data generated, as well the easy design for setting up experiments within the software.” If you would like to see Daniel’s full blogpost you can read it here.

Customers

Happy customers mean you have a product to be proud of. We are certainly very proud of the Progenesis QI and Progenesis QI for proteomics software. This blogpost entitled “What are our customers saying?”  highlights how our customers have used the software successfully in their day-to-day research. Comments like “with Progenesis QI for proteomics, we practically started getting publishable data within the first few hours.” from Dr. Lam Yun Wah at the Department of Biology and Chemistry, City University of Hong Kong and “Progenesis QI for proteomics provided me an excellent tool to profile hundreds of proteins with incredible precision to map proteins in cellular compartments of the photoreceptor cells of retina.” from Nikolai Skiba, Assistant Professor at the Albert Eye Research Institute, Duke University in the US, keeps us inspired knowing we are making a difference in people’s research.

More information

Learn more from the resources we have on our website. This useful summary, “Helping you to help yourself”, highlights all the valuable resource we have available, giving you the information you need to use the software more successfully. With user guides, a starter pack and FAQ’s, you can instantly get answers to the more common questions asked about the software.

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Progenesis QI for proteomics speeds up biopharmaceutical purification!

 

Most recombinant protein biopharmaceuticals are produced in specially designed expression systems typically using CHO (Chinese Hamster Ovary) cells. Many CHO proteins are simultaneously expressed along with high amounts of the desired biopharmaceutical, but they need to be removed by multi-step purification processes. Residual host cell proteins (HCPs) are low-level (1-100 ppm) process-related impurities that might be present in protein biopharmaceuticals even after extensive purification. HCPs could produce unwanted immunogenic responses in patients, they can reduce the efficacy or the stability of the drug or they can be responsible for drug degradation. For these reasons, the regulatory agencies required that all HCPs are identified and quantified prior to drug approval. The Biopharmaceutical industry relies on ELISA (enzyme-linked immunosorbent assays) for measuring the total HCP concentration expressed in ppm (or ng HCPs/mg biopharmaceutical). Mass spectrometry-based HCP analysis has emerged in recent years as a powerful alternative to ELISA [1-4] because it provides more extensive (proteome-wide) HCP coverage and is able to measure individual HCP levels.

Any LC-MS workflow for HCP analysis has three major goals: 1) identification of unknown HCPs; 2) reporting of the individual HCP quantification results expressed in ng HCP/mg biopharmaceutical (ppm concentrations); 3) monitoring of the HCP levels across multiple biopharmaceutical preparations. To accomplish these goals, two different LC/MS assays are required as illustrated by the workflow displayed in figure 1.

Figure 1. Workflow of the HCP analysis.

Figure 1. Workflow of the HCP analysis.

 

 

 

 

 

 

 

 

 

 

 

The Discovery HCP assay is performed in SONARTM mode in order to identify the unknown HCPs present in the purified biopharmaceutical and Progenesis QI for proteomics (QIP) is used for a proteome-wide database search to reveal the identity of these HCPs. For example, in the case of the NIST mAb, four HCPs and three spiked proteins (ADH, PHO and BSA) were identified as illustrated by the screenshot displayed in figure 2:

Table showing identification of 4 HCPs

Figure 2. Four HCPs (highlighted by red arrows) were identified by Progenesis QIP in the highly-purified NIST mAb.

A different type of LC-MS assay is required when multiple samples, produced from the bioprocessing of the same protein biotherapeutic, need to be analyzed with increased sample throughput, for the purpose of investigating HCP clearance. In this situation, the information gained from the HCP Discovery assay can be used to speed up the HCP identification and quantification process.

Using Progenesis QIP, the MS/MS fragmentation spectra of HCP peptides identified by SONAR acquisition can be assembled into spectral libraries, containing peptide precursors, charge states, retention times and relevant fragment ions. A list of HCP peptides sequenced from the NIST mAb is presented in Figure 3.

Table showing HCP peptides identified with a combination of NIST and SONAR

Figure 3. HCP peptides identified in the NIST mAb using SONAR acquisition.

 

 

 

 

 

 

 

 

 

 

 

The MS/MS fragmentation spectra of these peptides were assembled in a spectral library using Progenesis QIP. Peptides are sorted in the increasing order of their precursors. Two MS/MS spectra were recorded for four highlighted peptides, following fragmentation of their doubly and triply charged precursors.

Higher–throughput HCP Monitoring assays relying on 30 min peptide separations and employing MSE data acquisition are used for screening biopharmaceutical samples taken at every step of the purification process. The entire LC/MSE dataset is searched with Progenesis QIP against a spectral library for HCP identification, quantification, and monitoring.

To simulate an HCP monitoring assay, three protein digests standards (ADH–yeast alcohol dehydrogenase, BSA-bovine serum albumin and PHO-rabbit phosphorylase b) were spiked at four different concentration levels in four NIST mAb digests, while one protein digest (CLP_B-Ecoli chaperone protein) was spiked at the same concentration in all 4 samples. The LC/MSE data was searched in Progenesis QIP against a spectral library of 113 SONARTM fragmentation spectra of MIX-4 peptides (ADH, BSA, CLB-B, and PHO). Spiked proteins were easily tracked down to the lowest spiked levels (~ 20 ppm) across all five samples (20 LC/MSE runs) as exemplified by the graphs shown in Figure 4.

Graph showing measurement of spiked samples

Figure 4. (A) Example of protein level results obtained for the HCP Monitoring assay: the levels of spiked ADH were accurately measured in five NIST mAb samples; (B) Peptide level results of the HCP monitoring assay.

Eleven ADH peptides showed identical trends plots across all 20 runs. Four spiked samples, identified by letters A-D in this figure, containing different levels of ADH, BSA, PHO and CLP-B protein digests were spiked in the NIST mAb digest. The sample labeled “Blk” corresponded to the non-spiked NIST mAb digest. Each sample was analyzed with four replicates.

Protein measurements were obtained from multiple peptides and excellent correlation was obtained between the spiked and measured fold changes with RSDs under 10% for all measurements.

Progenesis QIP greatly simplifies the user interaction with HCP datasets. Extracting mass chromatograms and calculating peak areas for a multitude of peptide precursors (like the 113 peptides from the MIX-4 spectral library) can be a tedious process. In addition, the data from each individual sample replicates need to be compared in order to obtain the peptide level HCP trends. Finally, the HCP peptide level results need to be translated into HCP protein levels. All these steps are automated and they are performed rapidly in Progenesis QIP without significant user intervention. This saves a significant amount of time spent on data analysis, allowing for rapid results.

The experiment with spiked protein digests described above can be easily performed as a QC test to demonstrate the capability of the entire LC/MS platform to provide reliable HCP clearance results in a timely fashion.

Our collaborators from EMD Millipore asked us to test this capability for “real” mAb samples: they wanted to know which one of their four SCX (strong cation exchange) purification protocols produced “cleaner” purifications, with lower HCP content. The results are shown in Figure 5 and one of their protocols indeed worked better than the other three.

Graph showing monitoring of HCP peptides

Figure 5. Peptide level monitoring of three HCP peptides across five mAb preparations (one Protein A eluate and 4 SCX (strong cation exchange) chromatographic purifications using four different protocols (A-D). As illustrated here, Protocol D provided the best results.

Progenesis QIP allows purification laboratories to develop and test novel purification procedures in a relatively short time.

References:

  1. Doneanu CE, Anderson M, Williams BJ, Lauber MA, Chakraborty A, Chen W. Enhanced Detection of Low-Abundance Host-Cell Protein Impurities in High-Purity Monoclonal Antibodies Down to 1 ppm Using Ion Mobility Mass Spectrometry Coupled with Multidimensional Liquid Chromatography, Anal Chem, 2015, 87, 10283-10291.
  2. Huang L, Wang N, Mitchell CE, Brownlee T, Maple SR, De Felippis MR. A Novel Sample Preparation for Shotgun Proteomics Characterization of HCPs in Antibodies, Anal Chem, 2017, 89, 5436-5444.
  3. Weibin C, Doneanu CE, Lauber MA, Koza S, Prakash K, Stapels M, Fountain KJ. Improved Identification and Quantification of Host Cell Proteins (HCPs) in Biotherapeutics Using Liquid Chromatography-Mass Spectrometry, book chapter in Technologies for Therapeutic Monoclonal antibody characterization, Vol 3, ACS Symposium Series, 2015, 357-393.
  4. Doneanu C, Lennon S, Anderson M, Reah I, Ross M, Anderson S, Morns I, Yu YQ, Chakraborty A, Denbigh L, Chen W. A Comprehensive Approach for HCP Identification, Quantification and Monitoring Based on a Single Dimension (1D) LC Separation, Waters application note 720006262en, 2018.

Acknowledgments

Catalin Doneanu, Waters Corporations, Milford, MA, USA

What a ConFirenze! Explore. Dream. Discover.

When I heard that IMSC was in Florence I stuck my hand up to be on the exhibition booth.  It’s a city I’ve wanted to visit for years.

Ironically, we were so busy I didn’t get to look around!  Myself and my colleague, Mark Bennett, were attending for Nonlinear and, from the opening reception, we had a lot of interest in Progenesis.

This varied from researchers in nuclear physics to researchers analysing skin for anti-malarial research.  Progenesis really does cover a large breadth of scientific research, it’s strength being the ability to seek out differences without identification.

Quantify, then identify, is fundamental in the Progenesis workflow.

Mark Bennett demonstrating Progenesis to interested researchers Mark Bennett demonstrating Progenesis to interested researchers

In addition to busy ‘booth traffic’ and demonstrations, we had a workshop on the Thursday lunchtime.

Three speakers gave interesting accounts of how Progenesis QI and Progenesis QI for proteomics have helped them in their research:

Progenesis3 –Three personal accounts showing the power of Progenesis QI

  • Untargeted metabolomics using Progenesis QI for small molecules: Developing ion-chromatography-mass spectrometry for the investigation of cancer metabolism –James S.O. McCullagh, University of Oxford, UK
  • Metabolomic profiling of reactive metabolites in toxicology by MSE and Progenesis –Emilien Jamin, Toxalim (Research Centre in Food Toxicology) Toulouse university, INRA, France
  • Novel strategies for discovery of cardiovascular biomarkers in human plasma – Donald JL Jones, Leicester Cancer Research Centre, RKCSB, University of Leicester, UK

All three talks were engaging and we had good attendance.  The speakers had questions for each other and were engaged in conversation long after the workshop had finished.

Don Jones, Emilien Jamin and James McCullagh in conversation post workshop Don Jones, Emilien Jamin and James McCullagh in conversation post workshop

The workshop was so uplifting for me, it’s great when you hear customers picking out things that they really like in your product.

Don’t worry if you missed these presentations, we recorded the whole session, soon to be released!

If you’d like to be notified about these recordings, please email us and we’ll inform you when they become available.

So then it was on to the conference dinner, it completely exceeded expectations (which were not low).

We gathered at the Villa Viviani, located on the hill of Settignano, with a perfect view of Florence, which sits nestled in a natural bowl, just as the sun was setting.

The villa was home to Mark Twain for a time and one of my favourite quotes of his, “Explore. Dream. Discover.” seemed particularly apt for the conference and the wonderful evening.

It was so beautiful to stand there, listening to the live band and watching the dusk progress, an unforgettable memory.

Later, we were participants in an Italian birthday song game that had us up and down out of seats non-stop.  The next day my legs were really aching!

The sun setting over Florence at the gala dinner The sun setting over Florence at the gala dinner

Finally, we all dispersed home, the Progenesis researchers we spoke to heading back to their labs all over the globe.

It was a great Confirenze!!  Now I have to go back to Florence and see it properly, as a tourist.