Table of Contents

guest
2019-11-11
Tutorials
   教程 (中国语文)
     靶向 方法编辑
     靶向方法优化
     分析分类研究的数据
     处理已有定量实验数据
     小分子目标
     MS1 全扫描筛选
     靶向 MS/MS (PRM)
     非数据依赖型采集
     iRT 保留时间预测
   チュートリアル(日本語版)
     ターゲットメソッドの編集
     ターゲットメソッドの最適化
     によるグループ研究データの処理
     既存データ処理および定量実験
     によるターゲットメタボロミクス解析
     MS1フルスキャンフィルタ
     ターゲットMS/MS(PRM)
     DIA、データ非依存性解析
     iRT保持時間予測
   Targeted Method Editing
   Targeted Method Refinement
   Processing Grouped Study Data
   Existing and Quantitative Experiments
   MS1 Full-Scan Filtering
   Targeted MS/MS (PRM)
   Data Independent Acquisition
   Small Molecule Targets
   Small Molecule Method Development and CE Optimization
   Small Molecule Quantification
   Hi-Res Metabolomics
   Absolute Quantification
   Custom and Live Reports
   Advanced Peak Picking Models
   iRT Retention Time Prediction
   Ion Mobility Spectrum Filtering
   Collision Energy Optimization
   Spectral Library Explorer
   QuaSAR Quantitative Statistics
   ETH Targeted Proteomics Course Tutorials

Tutorials


[Chinese] [Japanese]

Try one of these tutorials, and get hands-on experience using Skyline with real data.

Introductory

 

Full-Scan Acquisition Data



 

Small Molecules

Small Molecule Quantification (23 pages, draft)
Hi-Res Metabolomics (13 pages, draft)


 

Advanced Topics

 

ETH Course Tutorials 2016 & 2018 (Skyline SRM/PRM/DIA + MSstats + mProphet tutorials with exercises)
SRM CourseETH Targeted Proteomics Course



教程 (中国语文)


[英语] [日语]

请选择尝试这些指南,以获得对实际数据运用Skyline软件的实际操作经验。

介绍性的

靶向方法编辑 (24 页)

 

靶向方法优化 (25 页)

 

小分子目标 (9 页)

 

 


全扫描采集数据

靶向 MS/MS (PRM) (39 页)

 



 


高级主题

 

更多即将推出!




靶向 方法编辑


获取针对靶向蛋白质组学实验创建Skyline文件的实际操作经验。在此指南中, 您将学会从pepXML和mzXML文件以及某个FASTA格式的背景蛋白质组文件中创建一个MS/MS谱图库。您会将这些信息与在GPM数据库网站(Gobal Proteome Machine)上的某个公共MS/MS谱图库相结合,以指导您创建一个新的Skyline文件去靶向分析一些特定的酵母蛋白质、肽段和子离子。从这个Skyline文件中,您会导出一个母离子-子离子对(transition)的列表,可以直接支持在AB公司的4000 Q Trap型质谱仪上检测分析 (24 页)。

[下载]

* - Skyline v0.6版本中开始引入, 已更新至 v1.4版本, 修订版本号为v2.5。




靶向方法优化


本教程是关于Skyline SRM数据分析的,内容涵盖导入仪器原始数据和对文档进行优化。利用多批SRM检测数据,本教程能够帮助你获得很多实际的操作经验。一开始,未优化文档给出的实验设计中,包括超过2000个需要检测的离子对,在Thermo TSQ平台上需要进行39次未预定的单次分析。在导入39次单次分析结果后,利用疏水性进行色谱保留时间回归分析,然后进行MS/MS图谱库的峰强度相关性分析,以提高检测到的色谱峰的可信度。在此基础上,可以利用Skyline进行实验设计优化,仅保留那些高可信度的色谱峰,从而筛选出精简的离子对列表。在优化完成后,对于预定实验设计,Skyline给出的离子对列表可以在一次分析中完成检测。在本教程中,这个实验设计的过程已经完成,你可以导入和查看多次重复实验检测的结果,以体验Skyline优化实验设计的效果。本教程总共有25页

[下载]

* - Skyline v0.6版本中开始引入, 已更新至 v1.4版本。

另外, 更多内容请参考我们发表在Proteomics 上的文章。(请引用)

The development of selected reaction monitoring methods for targeted proteomics via empirical refinement
[摘要]

 

了解更多 关于结果检查和优化实验设计方法的内容,可以阅读我们 ASMS 2009 的海报。

ASMS 2009 Poster




分析分类研究的数据


学习如何利用skyline有效的处理在一个生理状态下多个生物样品的实验数据。你将会使用一个可以被检测到的目标列表,并进一步优化该列表,使其可以只包含在健康和生病状态下在老鼠 (14只)的血浆中具有不同浓度的分子。在这个过程中,你讲学会如何使用skyline快速的研究和理解反常数据。你也会得到一些关于如何使用Skyline (版本3.1)来比较不同生理状态的经验。本教程总共有70页。)

[下载]

* - 根据版本3.1编写的使用说明




处理已有定量实验数据


通过本教程,你将学习如何处理已有的定量实验数据(不是由Skyline完成实验设计),包括利用稳定同位素标记肽段作为参考的实验。利用已知的离子对列表和已发布的SRM数据集,你可以获得这方面的实际操作经验,学习如何利用Skyline提供的色谱峰和色谱保留时间分析图表来进行高效的数据分析(本教程共39页)。 

[下载]

* - Skyline v0.7 版中引入, 更新至 v1.4版本。




小分子目标


学习如何利用skyline来分析小分子目标。你将会在skyline中导入一个小分子离子对的列表和14个Waters Xeve TQS 的实验数据。开始学习如何把skyline应用到小分子的实验中。本教程总共有9页。

[下载]

* - 根据版本3.1编写的使用说明




MS1 全扫描筛选


本教程将介绍如何利用Skyline完成无标记定量数据分析。在数据依赖(DDA)实验中,一般利用MS1信号强度构建离子流色谱峰(XIC),通过对比不同LC-Run中肽段的XIC面积来实现差异定量分析。在本教程中,你将学习利用Skyline从探索性实验数据集中构建图谱库,配置MS1过滤的Skyline文档,导入质谱仪原始数据文件,从MS1扫描中提取母离子的信号强度以构建XIC,然后根据MS/MS图谱肽段鉴定信息挑选合适的色谱峰,完成肽段定量。更重要的是,你还将学习如何利用Skyline进一步处理得到的定量结果。如果你对探索性实验无标记定量分析比较感兴趣,本教程将帮助你认识一个新的研究工具(本教程共37页)。 

[下载]

* - 在 v1.2版本中引入, 更新至v1.4版本, 修订版本号为v2.5。

另外, 关于Skyline无标记定量的算法和工作流程,更多内容参考我们发表在Molecular Cellular Proteomics 上的文章(请引用该文章):
Platform independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline. Application to protein acetylation and phosphorylation
[摘要]




靶向 MS/MS (PRM)


本教程将介绍利用Skyline完成高Agilent 6520 Q-TOF)低(Thermo LTQ)两种分辨率质谱平台的靶向MS/MS数据(也称为并行反应离子监测,PRM)分析,以便认识母离子-碎片离子对监测的肽段定量策略,在不同分辨率下选择性和灵敏度的差异。通过该教程,你将发现利用Skyline丰富的数据分析功能,能够帮助你理解自己的质谱数据,发现你自己质谱平台使用的新方法,以便更好地进行基于色谱分离的定量蛋白质组学分析。(本教程共39页) 

[下载]

* - 在v1.2版中引入, 更新至v1.4版, 修订版本号是v2.5。

另外, 更多信息请参考我们发表在Journal of Proteome Research 上的文章(请引用该文章)
Label-Free Quantitation of Protein Modifications by Pseudo-Selected Reaction Monitoring with Internal Reference Peptides
[摘要]




非数据依赖型采集


通过一个含有非依赖型数据采集和依赖型数据采集 (在同一个仪器上采集这两种数据)的实验方法来得到分析非依赖型数据的经验。定义和导出一个非依赖型数据采集方法的隔离方案。在进行非依赖型数据采集之前,进行依赖型数据采集,并利用其结果来建立一个质谱谱图库。根据质谱谱图库来选择对应目标蛋白质的多肽和离子对。用skyline导入并分析相关的非依赖型数据采集的结果来熟悉这个实验流程。本教程总共有40页。

[下载]

* - 根据版本2.6编写的使用说明




iRT 保留时间预测


本教程将引导你获得iRT技术的实际使用经验。iRT技术将校正过的、经验检测的肽段色谱保留时间存入一个库中,然后在预定实验中,用于离子对检测时间的确定和检测结果的验证。本教程中,你将学习如何校正自己的iRT计算器,以及更多的关于iRT-C18校正的过程。其中,iRT-C18是Biognosys在iRT-Kit中使用的肽段标准样品。本教程将引导你利用SRM数据、图谱库和探索性实验MS1过滤得到的色谱峰校正一个新的iRT计算器。同时,你将学习怎样对该iRT计算器进行再校正,以便用于洗脱梯度或者色谱柱不同的预定SRM实验。本教程将展示iRT预测的精度,以及高精度的iRT预测在色谱峰鉴定结果验证中,可以提供更高的可信度。(本教程共30页) 

[下载]

* - 在v1.2版本中引入, 更新至 v1.4版。

另外, 更多细节请参考我们发表在Proteomics上的文章 (请引用)
Using iRT, a normalized retention time for more targeted measurement of peptides
[摘要]




チュートリアル(日本語版)


[英語] [中国語]

これらのチュートリアルでは,実際のデータを用いた解析をご自身の環境で経験いただくことができます。

入門編


 

フルスキャン測定データの解析


上級者向け

iRT保持時間予測 (35ページ)

他の内容は近日公開する予定です!




ターゲットメソッドの編集


ターゲットとするプロテオミックスの実験に適したSkylineドキュメントを作成する為に必要な体験学習をする事ができます。このチュートリアルでは、pepXMLとmzXMLファイルからスペクトルライブラリを作成する方法、そしてFASTAファイルからバックグラウンドプロテオームファイルを作成する方法を学びます。これらの作成したファイルにGPM (Global Proteome Machine) 公開MS/MSスペクトルライブラリを追加することによって、選択された酵母タンパク質やペプチドそしてプロダクトイオンをターゲットするように Skylineドキュメントを更新する方法も学びます。この作成されたSkylineドキュメントからトランジションリストをエクスポートでき、AB 4000 Q Trapに使うことができます。(28ページ)

[ダウンロード]

* - Skyline v0.6にて導入。v1.4、v2.5で更新。




ターゲットメソッドの最適化


ここでは、幅広いSRMメソッドで測定されたデータをインポートし、Skylineを通じてトランジッションリストを最適化していく過程を体験することができます。ThermoのTSQでスケジュール化せず、2000を超えるトランジッションで39回の注入により測定した最適化されていないドキュメントから作業を開始していきます。まずは、39個のすべての測定データを一つの分析としてどのようにデータをインポートするかを学びます。ペプチドの疎水性度による保持時間予測やMS/MSスペクトルライブラリのピーク強度の相関性を利用して測定するピークの信頼度を高めていきます。Skylineの最適化ダイアログを使用して、最も信頼性のあるピーク以外を除外していきます。このようなステップにより、一回の注入による測定できるようにトランジッションのリストを減らしていきます。そして、一回の測定メソッドで複数の繰り返し測定の結果をインポートし確認します。(27ページ)

[ダウンロード]

* - Skyline v0.6より導入。v1.4にて更新。

私たちのProteomics誌の論文もご覧ください。(こちらを引用下さい)
The development of selected reaction monitoring methods for targeted proteomics via empirical refinement
[要約]


結果の確認やメソッドの最適化については、こちらのASMS2009のポスターもご覧ください。

ASMS 2009 Poster



によるグループ研究データの処理


大規模なデータの解析をSkylineにより効果的に処理する方法を学びます。ここでは、14匹のラットから採取した血漿での健常群と疾患群で検出したターゲットタンパク質の差がきちんと捉えられるようにしていきます。一連のデータを処理することを通じ、短時間でデータ全体を捉え、差を理解ができるSkylineの便利な画面表示を知ることができます。さらに、Skylineのバージョン3.1で導入された、Skylineによる群間の差の比較方法についても経験できます。 (73 ページ)

[download]

* - Skyline v3.1にて導入。




既存データ処理および定量実験


ここでは、公開されているSRMのトランジッションのリストと質量分析装置の測定データを用いて、Skylineにより安定同位体標識された内標準ペプチドを利用した定量分析について体験していきます。Skylineのピークエリアと保持時間のサマリーチャートを活用することで、あなた自身のデータを効果的に分析する方法を学びます。(45ページ)

[ダウンロード]

* - Skyline v0.7より導入。v1.4、v2.5にて更新。




によるターゲットメタボロミクス解析


ここでは、Skylineでの低分子化合物の解析について学びます。メタボロミクス研究で使用する低分子化合物のトランジションのリストとWaters社のXevo TQSで測定した14個のデータを使いながら、Skylineでどのように解析を行うかを習得していきます。(10 ページ)

[download]

* - Skyline v3.1にて導入。




MS1フルスキャンフィルタ


ここでは、ペプチドの発現量の差を、data dependent 測定(DDA)の実験でのMS1フィルタリングから得るための、Skylineドキュメントの作成方法について体験していきます。このチュートリアルでは、まず、探索的な実験のデータ(discovery data)からスペクトルライブラリを構築します。そして、SkylineドキュメントをMS1フィルタリング用に設定します。次に、測定したMS1スキャンデータからプリカーサーイオンのマスクロマトグラムを抽出するために質量分析装置で測定した生のデータをインポートします。クロマトグラム上の目的のピークのピッキングについては、MS/MSによるペプチド同定情報に基づき行われ、さらにSkyline上での処理を行うことで定量テータを得ることができます。もし、あなたが、探索実験のデータを用いたラベルフリーでの定量実験に興味を持っていらっしゃるなら、このチュートリアルはあなたの新しいツールとなるでしょう。(42ページ)

[ダウンロード]

* - Skyline v1.2より導入。v1.4、v2.5にて更新。

私たちのMolecular Cellular Proteomics誌の論文もご覧ください。(こちらを引用下さい)
Platform independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline. Application to protein acetylation and phosphorylation
[要約]




ターゲットMS/MS(PRM)


ここでは、ターゲットMS/MS(または、Parallel Reaction Monitoring(PRM、並列反応モニタリング))の方法について、低分解の質量分析装置であるThermoのLTQと高分解能の質量分析装置であるAgilent 6520 Q-TOFのデータを使って体験していきましょう。プリカーサーおよびフラグメントイオンを利用したペプチドの定量分析における選択性と感度について、分解能の異なる装置での測定による違いについて、理解することができるでしょう。クロマトグラフィーをベースとした定量プロテオミクスを実施する中で、Skylineが提供するさまざまな機能を活用することにより、質量分析データの理解と活用のための新しい方法を発見してください。(43ページ)

[ダウンロード]

* - Skyline v1.2より導入。v1.4、v2.5にて更新。

私たちのJournal of Proteome Research誌の論文もご覧ください。(こちらを引用下さい)
Label-Free Quantitation of Protein Modifications by Pseudo-Selected Reaction Monitoring with Internal Reference Peptides
[要約]




DIA、データ非依存性解析


ここでは、Data-independent acquisition(DIA、データ非依存性解析)について、同一装置で取得したDIAとDDAの測定データを利用していく方法で学んでいきます。まずは、SkylineでDIAデータを処理するための「Isolationスキーム」(DIAでのプリカーサーイオンのウィンドウ幅)の設定を行い、メソッドへエキスポートします。また、実験で使用するスペクトルライブラリをDDAのデータから、DIAの測定データの取得前に作成しておきます。そして、そのスペクトルライブラリからターゲットタンパク質を分析するためのペプチドやトランジションを選択します。そして、関連するDIAのデータをSkylineにインポートし、解析することで、DIAでの作業を始めるための基本的な流れを習熟していきます。 (43 ページ)

[download]

* - Skyline v2.6にて導入。




iRT保持時間予測


ここでは、スケジュール化された測定やピーク同定のための保持時間予測を行うためのiRT値について学んでいきます。校正された実測のペプチドの保持時間をライブラリーに蓄積することで、将来的に活用することができます。このチュートリアルでは、iRTカリキュレータによる校正方法や、Biognosys社により提案されているiRT-C18というキャリブレーションについて、iRT-Kitのペプチド標準品を用いて詳細に学んでいきます。さらに、SRMの測定データやスペクトルライブラリ、また、探索実験におけるMS1スキャンの測定データから得られたクロマトグラムピークからもiRT値を校正していきます。さらに、新しく設定したグラジエント条件で、新しいカラムを使用してスケジュール化したSRM測定をする際の、iRT値の再校正の方法も学習していきます。また、これらを通じて、iRT値に基づいて、保持時間を正確に予測することにより、クロマトグラムのピーク同定における信頼性が向上することもわかるでしょう。(35ページ)

[ダウンロード]

* - Skyline v1.2より導入。v1.4、v2.5にて更新。

私たちのProteomics誌の論文もご覧ください。(こちらを引用下さい)
Using iRT, a normalized retention time for more targeted measurement of peptides
[要約]




Targeted Method Editing


Get hands-on experience creating a Skyline document for a targeted proteomics experiment. In this tutorial, you will create a MS/MS spectral library from pepXML and mzXML and a background proteome file from a FASTA format file. You will combine these with a public MS/MS spectral library from the Global Proteome Machine to guide creation of a new Skyline document targeting selected yeast proteins, peptides and product ions. From this document, you will export a transition list, ready to run on a AB 4000 Q Trap. (26 pages)

[download]

* - written on Skyline v0.6, updated for v1.4, updated for v2.5, updated for v3.7

On February 10th, 2015, the Skyline Team produced Webinar #4: Targeted Method Design with Skyline, another great resource for this foundation topic.
[webinar]




Targeted Method Refinement


Get hands-on experience starting with a broad set of SRM measurements, importing instrument data, and refining the document. Start with an unrefined document requiring over 2000 transitions and 39 injections to measure unscheduled on a Thermo TSQ. Learn how to import all 39 injections into a single replicate. Use a hydrophobicity to retention time regression and ms/ms spectral library peak intensity correlation to improve confidence in measured peaks. Use the Skyline refinement dialog to remove all but the best transitions for the highest confidence peaks. Step through the scheduling process to reduce the document to a transition list that can be measured in a single injection. Import and view multiple replicates of the single-injection method. (26 pages)

[download]

* - written on Skyline v0.6, updated for v1.4, updated for v3.7

Also, see our paper in Proteomics (please cite)
The development of selected reaction monitoring methods for targeted proteomics via empirical refinement
[abstract]

On March 10th, 2015, the Skyline Team produced Webinar #5: Targeted Method Refinement with Skyline, another great resource for this topic.
[webinar]


Learn more about reviewing results and refining your methods by reading the ASMS 2009 poster.

ASMS 2009 Poster



Processing Grouped Study Data


Learn how to process multi-replicate study date effectively with Skyline. You will take an initial set of targets already refined as detectable and further refine this set for evidence of differential abundance between healthy and diseased subjects a study of plasma from 14 rats. In the process you will learn to use the powerful Skyline interactive displays to quickly investigate and understand data anomalies. You will also gain experience with the Skyline Group Comparison framework introduced in Skyline version 3.1. (70 pages)

[download]

* - written on v3.1

On April 7th, 2015, the Skyline Team produced Webinar #6: Effective Data Processing and Interrogation with Skyline, another great resource learning this material.
[webinar]




Existing and Quantitative Experiments


Get hands-on experience working with quantitative experiments and isotope labeled reference peptides, by starting with experiments with published transition lists and SRM mass spectrometer data. Learn effective ways of analyzing your data in Skyline using several of the available peak area and retention time summary charts. (41 pages)

[download]

* - written on Skyline v0.7, updated for v1.4

On December 1, 2015, the Skyline Team produced Webinar #12: Isotope Labeled Standards in Skyline, another great resource for this topic.
[webinar]




MS1 Full-Scan Filtering


Get hands-on experience creating a Skyline document to measure quantitative differences in peptide expression using the MS1 scans from your data dependent acquisition (DDA) experiments. In this tutorial, you will generate a spectral library from a discovery data set, set up a Skyline document for MS1 filtering, import raw mass spectrometer data to extract precursor ion chromatograms from MS1 scans, with peak picking guided by MS/MS peptide identifications, and further process the resulting quantitative data in Skyline. If you are interested in label-free quantitative analysis of discovery data sets, this tutorial will give you a new tool set for your investigation. (38 pages)

[download]

* - written on v1.2, updated for v1.4, revised for v2.5

Also, see our paper in Molecular Cellular Proteomics (please cite)
Platform independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline. Application to protein acetylation and phosphorylation
[abstract]

On October 21st, 2014, the Skyline Team produced Webinar #1: Getting the Most Out of DDA Data with Skyline, another great resource for working with DDA data in Skyline.
[webinar 1]
For an in-depth discussion peptide modifications, see Webinar #10: Working with Modifications in Skyline which was produced on September 29, 2016 by the Skyline Team and features importing PTMs, isotope labeling and importing large assay libraries, among other topics.
[webinar 10]

If you run into trouble seeing ID annotations in your chromatograms, be sure to consult Tip: ID Annotations Missing with Mascot Search Results. Even if you did not use Mascot, this tip may contain useful information.
[tip]




Targeted MS/MS (PRM)


Get hands-on experience working with targeted MS/MS (also referred to as parallel reaction monitoring - PRM) data acquired on a low resolution Thermo LTQ and a high resolution Agilent 6520 Q-TOF. Gain new understanding of the selectivity and sensitivity differences between peptide quantification using precursor and fragment ions measured on low and high resolution instruments. Discover new ways to work with and understand your own mass spectrometry data using the rich feature set provided by Skyline for working with chromatography-based quantitative proteomics. (40 pages)

[download]

* - written on v1.2, updated for v1.4, revised for v2.5

Also, see our paper in Journal of Proteome Research (please cite)
Label-Free Quantitation of Protein Modifications by Pseudo-Selected Reaction Monitoring with Internal Reference Peptides
[abstract]

On January 13th, 2015, the Skyline Team produced Webinar #3: PRM Targeted Proteomics Using Full-Scan MS and Skyline, another great resource for producing and working with PRM data in Skyline.
[webinar 3]
For a more advanced discussion see Webinar #9: PRM for PTM Studies which was produced by the Skyline Team on July 21, 2015 and covered using PRM data from a study of modifications on histones.
[webinar 9]




Data Independent Acquisition


Get hands-on experience working with data independent acquisition (DIA) data, using a workflow that utilizes DIA and DDA runs acquired on the same instrument in series. Define and export a DIA isolation scheme. Build a spectral library from DDA data acquired before the DIA runs for the experiment. Choose peptides and transitions for a target set of proteins based on the spectral library. Import and analyze related DIA runs in Skyline to master a simple workflow for beginning to work with DIA. (40 pages)

[download]

* - written on v2.6

On November 18th, 2014, the Skyline Team produced Webinar #2: Jump Start DIA Analysis with DDA Data in Skyline, another great resource for working with DIA data in Skyline.
[webinar]

On January 25, 2017 the Skyline Team produced Webinar #14: Large Scale DIA with Skyline, which highlighted the additional research and workflows developed in the 28 months since our first DIA webinar.
[webinar]




Small Molecule Targets


Learn how to target non-proteomic small molecule ions with Skyline. You will import a small molecule transition list used in a metabolomics experiment and import 14 runs from a Waters Xevo TQS. Start learning how to apply the power of the Skyline interface for small molecule experiments.(9 pages)

[download]

* - Originally written for Skyline 3.1; Updated for Skyline 19.1

On February 10th, 2015, the Skyline Team produced Webinar #4: Targeted Method Editing with Skyline, with a sneak peak of small molecule support.
[webinar]

On November 7th, 2017, the Skyline Team produced Webinar #16: Small Molecule Research with Skyline, with new material on small molecule support.
[webinar]

 

Learn more about using Skyline for small molecule quantification by reading this MSACL 2015 poster.

MSACL 2015 Poster




Small Molecule Method Development and CE Optimization


Learn how how to create a Skyline document that targets stable isotope labeled small molecules from a literature citation, specified as only precursor m/z, product ion m/z, and collision energy values. Perform retention time scheduling and collision energy optimization for small molecules by importing a multi-replicate data set from a Waters Xevo TQ-S using initial CE values from a Sciex triple quad. Learn how many existing Skyline features created initially for targeted proteomics use can now be applied to small molecule data.(28 pages)

[download]

* - Originally written for Skyline 4.1, updated for 19.1

On February 10th, 2015, the Skyline Team produced Webinar #4: Targeted Method Editing with Skyline, with a sneak peak of small molecule support.
[webinar]

On November 7th, 2017, the Skyline Team produced Webinar #16: Small Molecule Research with Skyline, with new material on small molecule support.
[webinar]




Small Molecule Quantification


Learn how to create a Skyline document that targets small molecules specified as precursor ion chemical formulas and adducts, and product ion m/z values. Import a multi-replicate data set collected using LC-MS/MS on a triple quadulpole, and see how many existing Skyline features created initially for targeted proteomics use can now be applied to small molecule data.(23 pages)

[download]

* - Originally written for Skyline 4.1, updated for 19.1

On February 10th, 2015, the Skyline Team produced Webinar #4: Targeted Method Editing with Skyline, with a sneak peak of small molecule support.
[webinar]

On November 7th, 2017, the Skyline Team produced Webinar #16: Small Molecule Research with Skyline, with new material on small molecule support.
[webinar]




Hi-Res Metabolomics


Learn how to create a Skyline document that targets small molecules specified as precursor ion chemical formulas and adducts. Import a multi-replicate data set collected on a Q Exactive Orbitrap mass spectrometer for a set of plasma samples, and see how many existing Skyline features created initially for targeted proteomics use can be applied to small molecule data. 

[download]

* - originally written for Skyline v4.1, updated for 19.1




Absolute Quantification


Get hands-on experience using Skyline with Excel to estimate the absolute molecular quantities of peptides in your experiments. (15 pages)

[download]

* - written on Skyline v1.1, updated for v1.4, updated for calibration features in v3.5

On December 1, 2015, the Skyline Team produced Webinar #12: Isotope Labeled Standards in Skyline, another great resource for this topic.
[webinar]

On April 15, 2016, the Skyline Team produced Webinar #13: Calibrated Quantification with Skyline, another great resource for this topic.
[webinar]

Also, see our paper in Nature Methods
Rapid empirical discovery of optimal peptides for targeted proteomics
[abstract]




Custom and Live Reports


Get hands-on experience working with the power of Skyline custom Live Reports to view, edit and export a wide range of values from your Skyline documents. These reports are perfect for use in Excel or with custom code written in R, Matlab, Java, C++ and other languages for doing deep statistical analysis after processing your instrument output with Skyline. Also learn to use the Skyline Results Grid view to gain access to these values and to add custom annotations while inspecting your data in Skyline. Follow this tutorial to greatly increase the scope of experiments you can achieve with Skyline. (38 pages)

[download]

* - written on Skyline v0.6, updated for v1.4, revised for v2.5




Advanced Peak Picking Models


Learn more about creating and testing advanced models for matching target peptides with chromatogram peaks in Skyline. With the 2.5 release, Skyline now supports creating linear combinations of individual peak scores using the mProphet semi-supervised learning algorithm.  In this tutorial, you will learn to generate decoy peptides and transitions, create and assess mProphet scoring models, and apply them to Skyline chromatogram peak picking, both for SRM and DIA/SWATH data.  You will learn about mProphet assigned q values (adjusted p values, based on FDR) and how you can associate them with your picked peaks and export them in a custom report. (28 pages)

[download]
 

* - written on Skyline v2.5

On January 25, 2017, the Skyline Team produced Webinar #14: Large Scale DIA with Skyline which included a fairly lengthy section on current peak picking strategies in DIA.
[webinar]

On April 4, 2017, the Skyline Team produced Webinar #15: Optimizing Large Scale DIA with Skyline which included more details on peak picking strategies in DIA.
[webinar]


The ETH DIA/SWATH course Tutorial 2 - Skyline DIA analysis also provides insight on using Skyline peak picking models for DIA with smaller target sets.
DIA/SWATH CourseETH DIA/SWATH Course

* - written on Skyline v4.1

You can also watch the DIA/SWATH Course presentation videos which include larger scale hands-on examples.




iRT Retention Time Prediction


Get hands-on experience with iRT, a technique for storing calibrated, empirically measured peptide retention times in a library for future use in retention time prediction for scheduled acquisition and peak identity validation. In this tutorial, you will calibrate your own iRT calculator and also learn more about the iRT-C18 calibration proposed by Biognosys for the peptide standards in their iRT-Kit. You will calibrate new iRT values from SRM data, a spectral library and chromatogram peaks filtered from MS1 scans in a discovery experiment. And, you will learn how to recalibrate these iRT values to schedule SRM acquisition on a new column with a new gradient. You will see how more accurate retention time prediction based on iRT can give you higher confidence in your chromatogram peak identity validation. (31 pages)

[download]

* - written on v1.2, updated for v1.4

On May 12, 2015, the Skyline Team produced Webinar #7: iRT Retention Time Prediction with Skyline, another great resource to learn more about iRT retention time normalization and library building concepts in Skyline.
[webinar]

On January 25, 2017, the Skyline Team produced Webinar #15: Large Scale DIA with Skyline with updated, advanced DIA workflows and using larger data sets. Webinar #14 also touches on iRT integration into method development.
[webinar]

Also, see our paper in Proteomics (please cite)
Using iRT, a normalized retention time for more targeted measurement of peptides
[abstract]




Ion Mobility Spectrum Filtering


Get hands-on experience using Skyline to work IMS-TOF data. Train a drift-time predictor for BSA spiked into yeast, and see how ion mobility separation improves the selectivity of chromagram extraction in complex data. Learn how to work with ion mobility data in Skyline and explore the 3D (m/z, IMS, intensity) spectra produced by IMS-enabled mass spectrometers.(26 pages)

[download]

* - written on Skyline v3.7

Also, see Brendan's presentation at the 2015 Agilent User Meeting at ASMS
Also, see Erin's presentation slides from the 2015 Skyline User Meeting at ASMS
Also, see the full data set on Panorama Public




Collision Energy Optimization


Get hands-on experience using Skyline to work with empirically measured optimal collision energy (CE) values. In this tutorial, you will create scheduled CE optimization transitions lists for a document with 30 peptide precursors. Using supplied RAW files from a Thermo TSQ Vantage, you will recalculate the linear equation used to calculate CE for that instrument. You will also export a transition list with CE values optimized separately for each transition. (12 pages)

[download]

* - written on Skyline v0.6, updated for v1.4

Also, see our paper in Analytical Chemistry (please cite)
Effect of Collision Energy Optimization on the Measurement of Peptides by Selected Reaction Monitoring (SRM) Mass Spectrometry
[abstract]




Spectral Library Explorer


Get hands-on experience working with the Skyline Spectral Library Explorer, new in v0.7. Learn more about working with isotope labels and product ion neutral losses using MS/MS spectral libraries containing 15N labeled and phosphorylated peptides. Use the Library Explorer to accelerate the transition between shotgun discovery experiments and targeted investigation. (22 pages)

[download]

* - written on Skyline v0.7, updated for v1.4




QuaSAR Quantitative Statistics


Preferred use of QuaSAR has changed from a GenePattern web page to a Skyline External Tool, installable and directly integrated into Skyline.

[download]




ETH Targeted Proteomics Course Tutorials


In 2016, members of the Aebersold lab at ETH, Zurich - with help from CRG, University of Washington, Purdue and Biognosys - presented the last week long course on targeted proteomics with SRM, PRM and DIA to 30 participants. During the course, the participants worked through 9 tutorials with follow-up exercises. This material has been made freely available on the Targeted Proteomics Course web site, providing a great resource to anyone interested in learning more about Skyline method editing and data processing (8 Skyline + MSstats + mProphet tutorials)

[go there] (2016)

Targeted Proteomics CourseETH Targeted Proteomics Course

* - written on Skyline v3.5

You can also watch the presentation videos.


In 2018, many of the same instructors with some new additions presented the second week long course on DIA/SWATH for proteomics to 50 participants. That course included new tutorials and lectures aimed at teaching DIA/SWATH data processing and use in proteomics research, with some very nice examples using Skyline. You can download them form the same location.

[go there] (2018)

DIA/SWATH CourseETH DIA/SWATH Course

* - written on Skyline v4.1

You can also watch the DIA/SWATH Course presentation videos.