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The hub for targeted proteomics
msoste 2013-11-13 10:19
Compared to its predecessors, the Skyline environment is conducive to high-throughput analysis, enabling the comparison of many targets over many different conditions with relative ease. It is quickly becoming the hub of targeted proteomics method development and analysis. Many existing external tools are already integrated into the software and new tools should be. I appreciate that the development team is actively staying ahead of the curve in proteomics so that when a new tool or technique is developed, Skyline is ready to implement it for all users.

Martin Soste
Picotti Lab
ETH Zurich
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Great User's meeting at ASMS 2012
Yishai 2012-06-04 04:35
Hello to the MacCoss lab,
Thank you for organizing the Skyline user's meeting. It was great to learn more about Skyline and have the opportunity to speak to other users.

We use Skyline on a daily basis and it has been a driving force for our SRM work and beyond.

Thank you,


Yishai Levin, PhD
Head of the Proteomics and Mass Spectrometry Unit
Weizmann Institute of Science
Rehovot, 76100, Israel
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Thank you
Alex Jones 2012-01-17 04:05
I have a good deal of experience of discovery proteomics but have only recently started designing MRM experiments. Skyline has let me efficiently use data from our Orbitrap (or colleagues datasets) to design and refine transitions. Quite simply Skyline has let me focus on my work rather than wrestling with software and different data formats. Brilliant work! Thank you and please keep it coming!

Alex Jones
The Sainsbury Laboratory.
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Skyline is great :)
ng.babaei 2011-08-24 18:33
Hi Brendan,
Thanks for this great software. I am PhD student with zero background in proteomics but using the online tutorial, I could easily learn and use skyline.Using Skyline also helped to get a very good knowledge in proteomics as well.
Thanks very much:-)
Negar, PhD candidate
Brien Holden Vision, UNSW
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Skyline = Game Changer
Peter Jackson 2011-08-17 14:22
Until I started using Skyline, new requests for protein quant and ID would require large amounts of time performing in-silico digests on prospector, copying/pasting into excel, and making my own templates for performing targeted experiments. Skyline is now a part of my regular pipeline and has allowed me to show people who might not be so mass-spec savvy, just how powerful targeted proteomics can be in their research.

I recommend it to everyone I encounter if they haven't already heard of it.

Peter Jackson
Associate Scientist - Analytical Physiology
Amyris Inc.
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Skyline has been indispensable
Thomas Neubert 2011-05-27 14:30
The scientists in my lab have been using Skyline for over a year, and we find it to be absolutely indispensable for the design, execution, and analysis of our SRM experiments. Skyline has many useful and innovative features, performs very well, and has allowed us to share and compare data with numerous labs using mass spectrometers made by several different manufacturers. Brendan MacLean has been extremely responsive to all questions and requests for new features. All in all we are delighted and very grateful for this fantastic software.

Thomas Neubert, Ph.D.
Associate Professor
Skirball Institute, NYU School of Medicine
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Disruptive Technology
Nathan Yates 2011-03-09 12:49
Skyline has been instrumental for the field of proteomics in that it provides a much needed research tool and, more importantly, a common framework that proteomics labs around the world can adopt and standardize to. The elegant design makes protein quantification a straightforward and efficient process, while the professional quality, publically available and freely distributed source code has driven up usage. What I find most exciting is that Skyline integrates mass spectrometry and biology, enabling the accurate and reliable measurement of specific proteins.

Nathan A. Yates, Ph.D.
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Angela Jackson 2011-03-08 18:22
Being that Skyline is vendor neutral, this powerful software is becoming a necessity for all our quantitative collaborations. We regularly work with suites of peptides synthesized with differing heavy labels and Skyline’s ability to assign modifications on a peptide-specific level is indispensible. PAR data is displayed directly in the peptide list allowing for easy identification of ions with interference. Skyline also took the laborious task of uploading a protein sequence, creating a scheduled MRM transition list, optimizing the CE and generating a final MRM method of the most intense ions and made it efficient! Skyline is truly an indispensible tool for targeted proteomic applications.

Angela Jackson
Research Associate
UVic GBC Proteomics Centre
Victoria, BC, Canada
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Skyline – I’m a fan
Sofia Waldemarson 2011-02-17 09:05
Skyline has from start been a great piece of software for analyzing MRM data. The continuous additions and rapid support from Brendan has made Skyline an invaluable platform for the entire MRM workflow. I appreciate how all data related to the analyses can be imported and directly compared and together with the tools available for method development making this process faster and easier. The visualization of spectra and chromatograms is Skyline’s major strength and aids in assessing the data. Skyline is also a great tool for explaining MRM data to students.

Sofia Waldemarson, PhD
Department of Immunotechnology
Lund University
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UI at its best
<1231> 2011-02-11 03:41
According to my experience this is the best software in terms of User Interface among all others which were designed by the companies in its class. The good thing about this software is that simple,robust and its FREE OF CHARGE . The ability to compare cross platforms is the paramount feature of this software.

Method development is spot on. When it comes to data analysis and quantification there is still some space to improve by looking from the company designed softwares.

I am more than happy using this software at my work.

Praveen Kumar,
PhD student,
EMA Universität Greifswald,
Department of Functional Genomics,
Greifswald, Germany.
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Skyline: great software tool
Karin Wolters 2011-02-09 03:12
Skyline provides a great interface for the development and analysis of your MRM assays.
Best way to find out is to try it yourself! Just a few statements about it: it's really user-friendly; you get started easily; the tutorials clearly explain the various possibilities; MRM assays can be prepared for different mass spectrometers; the various (graphical) representations to visually inspect your results are great and you have multiple options to manually adjust settings; the support for questions and problems is really fast and helpful!

Karin Wolters Ph.D
Department of Analytical Chemistry
University of Groningen
The Netherlands
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Biomarker Development
Steve Pennington 2011-02-08 07:39
The messages and comments received so far say it all! We have been using Skyline for developing high quality MRM's in a range of biomarker discovery and development projects and it has become indispensable. The excellent features, most notably the ease with which we can use it to establish correlations between in-house spectral library data and MRM transitions as well as produce methods to undertake collision energy optimisation runs, and the great support from Brendan, have made a dramatic positive impact on our MRM work. In my experience it is rare indeed to find software that so superbly meets the needs of end-users.

Steve Pennington
Prof of Proteomics
UCD Conway Institute
University College Dublin
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Skyline: The ideal tool for quantification by SRM
Stephen Holman 2011-02-08 02:52
I have been using Skyline for several months now and find it an invaluable resource. Its functionality means that it easily surpasses all competitors in terms of ease of use, meaning that time can be spent performing meaningful experiments rather than laboriously building acquisition methods. The regular updates and improvements to the program means that it is becoming increasingly useful in the area of quantitative proteomics using SRM, and thus will continue to be the number one software in this field.

When I had a problem with Skyline, Brendan Maclean (the program's designer) was more than happy to work on altering the program to meet my needs, and did so in a timely manner. The implemented change has improved Skyline in the context of my work, and I am very grateful to Brendan for his help.

Stephen W. Holman Ph.D
Michael Barber Centre for Mass Spectrometry
Manchester Interdisciplinary Biocentre
The University of Manchester
M1 7DN
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Great software.
<1107> 2011-02-03 22:27
Skyline is a very user-friendly, intuitive software tool that significantly speeds up my MRM-MS analysis. I particularly appreciate its compatibility with various MS platforms from different vendors and ease-of-use which make performing MRM assays using Skyline a smooth experience. Video tutorials and exceptional tech support are yet another great thing about this software. The usefulness of Skyline for performing MRM experiments is without question. Additionally, I found Skyline to be a fast and robust tool for calculating m/z of tryptic peptides and their modified forms. I use this functionality on a regular basis when I work on projects which aim to look for PTMs using LC-MS/MS and MALDI-MS and, in my opinion, the flexibility of Skyline as a m/z calculator makes it a great replacement of some popular software tools already available which are much less flexible.

Pawel Sadowski, Ph.D.
Postdoc fellow and satisfied Skyline user, NYU Protein Analysis Facility
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Skyline is the ideal all-in-one solution for our targeted analysis of cell surface proteins
Thomas Bock 2011-01-31 01:18
In our laboratory we increasingly move towards the targeted analysis of cell surface proteins. With the integration of Skyline into our SRM workflows, I think we found an ideal all-in-one solution that combines a user friendly interface with the necessary flexibility that is needed in our research environment.
One of my preferred features is the easy generation and handling of large N glycopeptide spectral libraries that I further use for the selection and quality control of peptide transitions. The possibility to introduce a background proteome is great for additional protein information and to filter for proteotypic peptide sequences.
Skyline’s powerful data analysis capabilities are fantastic and its features for peak picking and integration are routinely used in our laboratory.
In addition, I highly profited from the excellent software support and the nice video tutorials helped me to being productive in a very short time after installation of the software. To me, Skyline is the optimal solution for anybody planning to do SRM measurements.

Thomas Bock, Diplombiologe
PhD-student, group of Dr. Bernd Wollscheid,
NCCR Neuro Center for Proteomics,
Institute of Molecular Systems Biology, ETH Zürich, Switzerland
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Skyline addresses a critical translational gap in our biomarker pipeline
Will Thompson 2011-01-13 11:13
Skyline directly addresses a critical gap in our biomarker proteomics pipeline – the translation from biomarker discovery (‘omic LC/MS/MS) to biomarker verification (targeted LC/MS/MS).
Skyline has proven to be a fast, multiplexed, and reliable tool for the creation of complex MRM methods. We utilize spectral libraries and Skyline to quickly generate preliminary methods for our Xevo TQ, which target tens to hundreds of peptides with many transitions for each peptide. The data generated from a single Skyline-directed MRM analysis then allows us to maximize sensitivity and duty cycle by identifying the 2 to 3 best transitions and the proper retention time window for scheduled monitoring of each peptide. With the recent improvements in peak smoothing and integration algorithms along with the customizable reports, we are now using Skyline to also confidently analyze our targeted proteomics data. Finally, the deftness with which the Skyline development team takes user recommendations and includes them in new revisions of the software package is truly impressive.

J. Will Thompson, Ph.D. and M. Arthur Moseley, Ph.D
Duke Proteomics Core Facility
Duke Institute for Genome Sciences and Policy
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Skyline, the best proteomics software period
Brett Phinney 2011-01-12 17:36
It would not be a stretch to say that Skyline not only is the best software available for targeted proteomics applications, it is the best proteomics software period. It is better than any commercial software I have ever used and has great support from Brendan. We use it all the time and could not live without it. The fact that it is open source and freely available is great and allows us to share our skyline data easily with our collaborators. Here are some of the things Skyline excels at; it fosters open collaboration and data sharing between labs and investigators, it is vendor neutral and handles dozens of data files with ease, it is powerful but intuitive and easy to use at the same time. I could go on and on. It is a real breakthrough for targeted proteomics

Brett S. Phinney, Ph D.
UC Davis Genome Center
Director Proteomics Core Facility
Twitter =
Office# 530-754-5298
Google Voice# 754-Brett-04
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Skyline is an essential tool for all mass spectrometry labs
Chris Wu 2011-01-10 13:57
I would recommend Skyline to anyone interested in quantitative mass spectrometry measurements, particularly for multiplexed SRM analyses. One of the first things that I assign to new members of my lab, students I am mentoring, or collaborators wanting to be involved in the experimental pipeline, are the Skyline tutorials. The tutorials are comprehensive and easy to understand. This is extremely effective as an educational tool, particularly for those who have minimal or no background in quantitative mass spectrometry. For the members of my lab who are actively performing SRM-MS analyses, Skyline has TRANSFORMED the entire analytical pipeline (including the painful process of setting up methods for scheduled multiplexed experiments) to something that is routine and turnkey. Before Skyline was available, the most difficult parts of the experiment were 1) building the method and 2) data analysis. Now, the most difficult parts of the experiment are sample preparation and peptide separations, as it should be. Finally, and most importantly, the support for this software suite is phenomenal. Response times are typically within a few hours, and new features are continuously being added, frequently in direct response to user suggestions. I have never experienced this type of support for commercial proteomics software, on which I have unfortunately spent thousands of dollars. This is an open source tool developed in an academic lab, and I really don’t know how they maintain this type of service. It really feels like a community effort. Members of my lab have frequently commented they really feel involved in the software design process. This type of interactive support is truly unique and enabling and yet, disappointingly under-appreciated.

Christine Wu, Ph.D.
Associate Professor of Cell Biology
University of Pittsburgh School of Medicine
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Skyline: An open source software that supports your SRM workflow from the start to the end
Christina Ludwig 2010-12-18 04:56
Skyline is my favorite software for SRM analysis, because I can use it throughout the whole SRM workflow: 1. starting with the design of SRM assays for all peptides/proteins of interest based on spectral libraries or on systematical filtering; 2. optimization and validation of these assays in a representative sample of interest; 3. analysis of the final SRM assay collection in high throughput; 4. quick quality control and comparison of all measurements; 5. and finally the in-depth quantitative manual analysis of the experiment. For my own work the most outstanding features of Skyline, compared to any other available SRM software, are the easy and straightforward import of spectral libraries from any source, the use of a background proteome, which provides a lot of important protein information and enables the choice of genetically unique peptides, and finally the support of any type of posttranslational modifications (in particular phosphorylation and neutral-loss transitions). In our institute triple quadrupole instruments from various different manufacturers are used on a regular basis, therefore I also very much appreciate the fact that Skyline can be applied to any data type and that methods, libraries, reports and results can very easily be shared.
The fast and competent support by the Skyline team is outstanding and I personally experienced the genuine implementation of my own suggestions and requests into Skyline. Hence I highly recommend the use of Skyline to everyone interested in doing SRM measurements!

Christina Ludwig, Ph.D.
Research scientist, group of Ruedi Aebersold
Institute of Molecular Systems Biology, ETH, Zürich
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Skyline: a necessary tool for evolving proteomics labs
Susan Abbatiello 2010-12-15 19:10
Skyline is a powerful software platform that enables any lab to easily create targeted SRM/MRM-MS methods from empirical data or in silico sources. It is continually adding features and improving the flexibility of MRM-MS method creation, data analysis and data reporting. The graphics included to monitor the quality of the MRM-MS data, such as peptide retention times and peak area coefficient of variation (CV) quickly enable the user to ensure the acquired data is at the level it needs to be. It is a simple yet multifaceted software package that delivers many options for various experimental designs including multiple peptide labeling strategies. I find the peak integration of MRM-MS data to be very reliable and simple to use, especially for large data sets. It is easy to learn and provides all of the tools necessary to design and execute a targeted, quantitative study of peptide and protein biomarker candidates on any MS vendor platform. Software support is excellent.

Susan Abbatiello, Ph.D.
Research Scientist, Proteomics Platform
The Broad Institute of MIT and Harvard
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Skyline: Everything you could ever ask for in a targeted proteomics software tool
Lisa Zimmerman 2010-12-09 09:36
In my opinion, the development of Skyline has transformed the way targeted proteomics experiments are performed within our laboratory. As a laboratory interested in discovery and verification of biomarkers, Skyline, a vendor neutral software, offers all the necessary capabilities to perform a MRM experiments from start to finish. The ability to create spectral libraries from existing tandem mass spectrometry data which can then be used to select the optimal proteotypic peptides and transitions allows for easy MRM method-building for a few or hundreds of proteins. The export features allow instrument methods to be generated for MS platforms for simple unscheduled methods, in addition to creating scheduled methods, allowing for increased MRM throughput. It doesn’t stop there! Skyline makes data processing and integration very easy allowing users to visual their data in various graphical forms. The ability to create custom-design report templates which can be shared offers the ability to standardize data reporting within or across laboratories. Skyline’s development and support team is absolutely amazing and always open for feedback on how to expand and improve the software’s current features to meet the demands of its users. Skyline is easy to use and the effective tutorials and videos created by the Skyline team will have you up and running MRM experiments in no time!

Lisa J. Zimmerman, Ph.D.
Research Associate Professor, Ayers Institute, Vanderbilt University
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Skyline: full support for easy MRM assay development and implementation
Birgit Schilling 2010-12-08 19:27
What I like about Skyline is that it is a fine open source software program that has become instrumental in integrating mass spectrometric discovery and verification workflows. I particularly appreciate that I can very easily incorporate spectral libraries obtained from our own MS/MS discovery experiments into Skyline to then very quickly and efficiently design targeted verification MRM assays. It is particularly nice that Skyline supports any type of stable isotope labeling as well as posttranslational modifications (i.e. phospho peptides, acetylated peptides, deglycosylated peptides, etc.) as we often target posttranslationally modified peptides in our MRM assays. I also like the fact that all acquired LC-MRM assays and data sets can be fully processed and integrated in Skyline, which becomes particularly important if one performs a large study as Skyline also helps to keep imported data files organized. We are using Skyline documents to send MRM assay templates and MRM data back and forth between collaborators. Last but not least, software support from Skyline designers is exceptional.

Birgit Schilling, Ph.D.
Gibson Laboratory, Buck Institute for Age Research, Novato, CA
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Skyline is a valuable tool for targeted proteomics
Jeff Whiteaker 2010-12-03 10:52
Our laboratory has found Skyline to be an instrumental tool in developing targeted mass spectrometry methods. Specifically, Skyline has enabled us to identify SRM/MRM transitions to hundreds of target peptides in an efficient manner, refine and optimize those transitions empirically, and share the results with collaborators. The ability to work with multiple vendors' platforms is particularly attractive for our laboratory, and this makes possible a single streamlined workflow for our targeted proteomics experiments. Support for the software has been fantastic. Tutorial videos and slidedecks get new users up and running in a timely manner, and the development team has been openly engaged in discussing and implementing new features.
Jeff Whiteaker, Ph.D.
Director of Proteomics, Paulovich Laboratory, Fred Hutchinson Cancer Research Center
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