Skyline Custom Reports and Results Grid

Transcription

1 Skyline Custm Reprts and Results Grid The Skyline Targeted Prtemics Envirnment prvides infrmative visual displays f the raw mass spectrmeter data yu imprt int yur Skyline dcuments. It allws yu t wrk with this data, t refine the peptides and transitins yu are measuring and t tune integratin bundaries. A rich variety f calculated values and statistics may be exprted frm yur Skyline dcument t a tabular cmma separated value (CSV) file perfect fr further analysis in tls like Excel and R. The Skyline Results Grid displays many f these values and allws yu t edit custm anntatins as yu wrk with yur data. The first part f this tutrial will fcus n Skyline custm reprts. Yu will learn hw they can be designed, shared and used t exprt raw values and summary statistics frm yur Skyline dcuments. Exprted reprts can be used in the fllwing ways: Deeper statistical analysis in tls like Excel r R Brader statistical analysis acrss multiple experiments and/r labratries Enfrcing instrument quality cntrl with summary statistics Prviding an input frmat fr ther custm build sftware tls Prviding an imprt frmat suited t relatinal data repsitries In develping Skyline we aim t create a vendr neutral platfrm fr targeted prtemics investigatin. Skyline can exprt similar custm reprts fr dcuments ppulated with the data frm Agilent, AB Sciex, Therm Scientific and Waters instruments. The ability t create cnsistent reprts acrss instrument platfrms greatly facilitates crss instrument cmparisns and large multi site studies. Understanding hw t create custm reprts, tailred t yur wn experiments, is critical and described in detail in this Skyline custm reprts tutrial. The secnd part f this tutrial will fcus n the Results Grid view in Skyline, which prvides real time access t many f the data fields available in the custm reprts. Yu will learn t custmize clumns in the Results Grid fr immediate access t imprtant values, as yu inspect and refine yur mass spectrmeter utput in Skyline. Yu will als learn t wrk with custm anntatins t prvide a cntrlled vcabulary fr further interpretatin f and human interactin with yur results, which can als be exprted in custm reprts. Getting Started T start this tutrial, dwnlad the fllwing ZIP file: 1

2 Extract the files in it t a flder n yur cmputer, like: C:\Users\brendanx\Dcuments This will create a new flder: C:\Users\brendanx\Dcuments\CustmReprts Nw pen the Study7_example.sky file in this new flder by duble clicking, r by using Open n the File menu in a running instance f Skyline. Data Overview The dataset in Study7_example.sky is a subset f a study that was perfrmed by the Clinical Prtemics Technlgy Assessment fr Cancer (CPTAC) cnsrtium (T. Addna et al Multi site assessment f the precisin and reprducibility f multiple reactin mnitring based measurements f prteins in plasma, Nature Bitechnlgy, 2009, 27, ). This particular study describes a calibratin curve at different cncentratins f analyte peptides and a cnstant spike in level f their crrespnding istpe labeled internal standards. The Skyline file displays data frm a subset f CPTAC Study 7.2 data (in the paper referred t as Study II ) acquired at ne CPTAC site. The Skyline file shws data fr 10 different peptide sequences mnitred by LC MRM MS with cncentratins fr the analyte prteins (see light peptides) ranging frm cncentratins at 500 fml t 2.92 fml (replicates J: 500 fml; I: 275 fml; H: 151 fml; G: 83 fml; F: 46 fml; E: 25 fml; and D: 8.55 fml lwer cncentratin pints althugh acquired are nt included in this tutrial). Heavy internal standard (IS) peptides are each spiked in at a cncentratin f 50 fml. T get a first impressin what this dataset lks like perfrm the fllwing steps: Click n the HGFLPR peptide t highlight it. On the View menu, chse Peak Areas, and then click Replicate Cmparisns. A new windw appears that shws peak area fr each replicate. The blue bars represent the heavy labeled versin f this peptide (always spiked in at 50 fml), and the red bars represent the light analyte at varying cncentratins (each cncentratin pint was acquired in technical quadruplicate). Other tutrials describe hw yu can view and edit dcuments like this ne. Fr the purpses f this tutrial, hwever, the Study7_example.sky dcument has received an initial integratin review, fully preparing if fr reprts t be exprted and used in deeper statistical analysis. Befre cntinuing with the rest f the tutrial: Clse the Peak Areas view. 2

3 Creating a Simple Custm Reprt T create yur first Skyline custm reprt template, perfrm the fllwing steps: On the File menu, chse Exprt, and click Reprt An Exprt Reprt frm will appear that will prbably list at least the Skyline default reprt templates shwn belw: Cntinue creating a new Skyline custm reprt template by perfrming the fllwing steps: Click the Edit list buttn. In the Edit Reprts frm, click the Add buttn. In Edit Reprt frm, enter Overview in the Reprt Name field. The frm shuld nw lk like: 3

4 Expand the field grups Peptides and Results by clicking n the + buttn t see many mre fields that can be exprted. See Windw belw. Frm the expanded Peptides grup select the Sequence field. Click the Add buttn t add the PeptideSequence field t the reprt template clumn list, shwn n the right side f the frm. The frm shuld nw lk like: 4

5 Nte that the fields in the Edit Reprt frm, shwn abve, have a distinct hierarchical structure. At the highest level are the prtein specific exprt fields which are shwn at the bttm f the left bx (PrteinName, PrteinDescriptin, PrteinSequence, PrteinNte, and the expanded Results grup abve these that cntains the general fields ReplicateName, ReplicatePath, FileName and SampleName). Skyline custm reprts als prvide peptide specific exprt fields, cntained in the expanded Peptides grup at the tp f the left bx, such as Sequence and AverageMeasuredRetentinTime amng thers. The peptide fields include a PeptideResults grup, which is nt expanded yet. Als unexpanded is the Precursrs grup, which cntains precursr specific exprt fields, a PrecursrsResults grup, and finally a Transitins field grup. The Transitins grup, in turn, cntains the transitin specific exprt fields and a crrespnding TransitinResults grup, cntaining the mst detailed results values, such as Retentin Time, Fwhm, StartTime, EndTime, Area and Backgrund, frm which many f the higher level results field values are derived. In the Edit Reprt frm cntinue t add fields t yur first custm reprt template by perfrming the fllwing steps: Expand the Precursrs grup (lcated directly belw the expanded Peptides grup at the very tp) by clicking n the + icn. Expand the PrecursrResults grup field nw visible in the expanded Precursr grup. 5

6 Select IstpeLabelType field (marked belw with a red dt) frm the expanded Precursrs grup. (If necessary, scrll dwn r resize the frm.) Click the Add buttn t add IstpeLabelType t the reprt template clumn list. Repeat this prcedure t add the fllwing additinal fields frm the PrecursrResults grup t the reprt template clumn list: BestRetentinTime the RetentinTime value f the transitin with the highest maximum intensity fr the particular precursr TtalArea the summed Area values f all individual transitins fr the particular precursr (fr mre infrmatin n field values, see Glssary) Check the Pivt Replicate Name check bx in the lwer left crner f the Edit Reprt frm. This shuld leave the Edit Reprt frm lking like: Click the Preview buttn in the upper right crner f the Edit Reprt frm. The Preview Reprt frm shuld appear lking like: 6

7 The Preview functin is quite helpful when designing reprt templates. At times it may even serve as a quick substitute fr exprting a reprt t a file, since yu can select all f the values in the Preview Reprt frm by clicking the upper left rectangle. Pressing Ctrl C, then cpies the full reprt. After which, it can be pasted int anther tl, like Excel. The reprt preview displayed in part abve, fr replicates 7_2_D_01, and 7_2_D_02, shws precursr BestRetentinTime and TtalArea fr all 10 peptides in bth their light and heavy frm acrss all LC MRM MS acquisitins/experiments in the clumns t the right. By scrlling t the right, yu can see clumns fr all 28 LC MRM MS acquisitins. Maximize the Preview Reprt frm by clicking n the square in its upper right crner t see mre clumns. In this example 7_2_D_01, and 7_2_D_02 are taken frm samples at the same light analyte cncentratin. Therefre, TtalArea values are expected t be similar. Each letter D thrugh J indicates a different light analyte cncentratin. Each cncentratin was acquired in technical quadruplicate, indicated by numbers 01 thrugh 04. The heavy peptide cncentratins were held cnstant at 50 fml in all samples. Clse the Preview Reprt frm by clicking n the x in the upper right crner t return t the Edit Reprt frm again. T save this reprt template: Click the OK buttn. 7

8 Yu will see the new Overview reprt template appear in the Reprt template list. Click the OK buttn again t return t the Exprt Reprt frm. The new Overview reprt template is nw added t the Skyline reprt template list. The Exprt Reprt frm shuld nw lk like: 8

9 Frm the Exprt Reprt frm yu can nw exprt yur new reprt by selecting Overview in the Reprt list and clicking the Exprt buttn. Fr nw, click the Cancel buttn, dismissing all pen frms, in rder t start the prcess frm the riginal Skyline dcument view. Exprting Reprt Data t a File T exprt the reprt yu created and previewed abve t a CSV file, d the fllwing: On the File menu, chse Exprt, and click Reprt. Select Overview in the Reprt list f the Exprt Reprt. Click the Exprt buttn. Enter Overview_Study7_example.csv in the File name field f the Exprt Reprt save frm. Click the Save buttn. If yu switch t a Windws Explrer windw, shwing the CustmReprts flder yu created fr this tutrial, yu will see the Overview_Study7_example.csv file yu just created. Open it nw with Excel and cnfirm that its cntents are quite similar t the preview yu displayed earlier. Nte that the file cntains the clumn header names, which yu cannt get by simply cpying and pasting frm the preview frm. Sharing Reprt Templates Reprt Templates in Skyline are very flexible, easy t manage and mdify, and share with cllabratrs. There are many reasns yu might want t share a custm reprt template, whether yu are participating in a large multi labratry study r wrking with a single cllabratr, whether yur are prviding the prtemics cmmunity with yur wn custm tl that depends n particular fields frm Skyline dcuments r prviding supplementary infrmatin with a manuscript t allw thers t repeat yur prtcls. The Skyline supprt fr instruments frm multiple vendrs means yur shared reprt templates can be used t gather data frm multiple sites with different instrumentatin. T share the Skyline reprt template yu created in this tutrial, perfrm the fllwing steps: On the File menu, chse Exprt, and click Reprt. Click the Share buttn in the Exprt Reprt frm. Skyline will present the Save Reprt Definitins frm, as shwn belw: 9

10 Check Overview in the reprt definitins list. Click the OK buttn. Enter Overview in the File name field f the Save As frm. Click the Save buttn. The reprt template is saved t the file Overview.skyr in the CustmReprts flder created fr this tutrial. Yu culd nw share it with yur cllabratrs, with yur wn analysis tl r as supplementary data fr a manuscript. Managing Reprt Templates in Skyline T gain an understanding f hw thers wuld use the custm reprt template yu have nw created and shared, yu need t first delete it frm yur system. Then yu can add it back using the shared reprt file Overview.skyr that yu created in the previus sectin, as if yu had acquired this shared template frm anther surce. The Exprt Reprt frm shuld still be pen after the steps perfrmed in the previus sectin. T delete the Overview reprt template frm yur Skyline settings, d the fllwing: Click the Edit List buttn. Select the Overview item in the Reprt list f the Edit Reprts frm. The Edit Reprts frm shuld nw lk like: 10

11 Yu can use the Up and Dwn buttns t change the rder f the items in the Reprt list. T mve the Overview item t the bttm f the list, d the fllwing: Click the Dwn buttn twice. Click the Up buttn three times t mve the Overview item t the tp. T remve the Overview item frm the list, d the fllwing: Click the Remve buttn. Click the OK buttn. The Exprt Reprts frm shuld nw lk like: 11

12 Just as when yu first brught it up in this tutrial. T demnstrate hw yur cllabratrs wuld imprt a new reprt template int Skyline fllw the steps described belw: Click the Imprt buttn. Select the Overview.skyr file yu created earlier in the Open frm (which shuld still be shwing the cntents f the CustmReprts flder fr this tutrial). Click the Open buttn. These actins shuld add an Overview item back t the Reprt list in the Exprt Reprt frm. T verify that this is indeed the reprt yu saved, perfrm the fllwing steps: Select the Overview item in the Reprt list. Click the Preview buttn. Skyline will present a Preview Reprt frm cntaining the same values yu saw earlier in this tutrial. Mdifying Existing Reprt Templates Yu can, f curse, create much mre data rich reprts than the simple Overview reprt yu first created in this tutrial. Yu can d this by creating anther new reprt template, by editing yur existing Overview reprt, r, as shwn belw, by making a cpy f the Overview reprt and editing the cpy. T create a new, mre cmplex reprt frm a cpy f the Overview reprt template, perfrm the fllwing steps: 12

13 Click n Edit List buttn in the Exprt Reprt frm. Select the Overview item in the Reprt list. Click n the Cpy buttn. Skyline will present the Edit Reprt frm with the fields frm the Overview reprt template already added, as shwn belw: The reprt name fr this new reprt template has nt yet been assigned. T assign it nw: Enter Study 7 in the Reprt Name field. Cntinue t add mre fields as described belw by selecting each field and clicking the Add buttn in the middle t add each field t the clumn list fr yur reprt template, n the right side f the Edit Reprt frm: Expand the field grups Peptides and Results by clicking the + icns t their left. Frm the expanded Results grup, add the fllwing fields: FileName the name f the file frm which the data was imprted SampleName the sample name, if the data was imprted frm a multi sample WIFF file, r the file name again, if nt ReplicateName the replicate name assigned t the data during imprt 13

14 Frm the tp level add PrteinName. Frm the expanded Peptide grup, add the AverageMeasuredRetentinTime field the average retentin time ver all replicates. Expand the PeptideResults grup. Frm the expanded PeptideResults grup, add the fllwing fields: PeptideRetentinTime peptide RT fr each replicate run RatiTStandard area rati f light t heavy Expand the Precursrs grup. Frm the expanded Precursrs grup, add the fllwing fields: Charge (displayed in the clumn list as PrecursrCharge) Mz (displayed in the clumn list as PrecursrMz ) Expand the Transitins grup. Frm the expanded Transitins grup, add the fllwing fields: PrductCharge PrductMz FragmentIn the fragment in name (e.g. y8, y10, b7, etc.) Expand the PrecursrResults grup. Frm the expanded PrecursrResults grup, add the fllwing fields: MaxFhwm the maximum full width at half max (FWHM) f the transitins fr the particular precursr MinStartTime elutin time start at the baseline f a peak, which will be the same fr all transitins (Q1/Q3) frm the same precursr MaxEndTime elutin time start at the baseline f a peak, which will be the same fr all transitins (Q1/Q3) frm the same precursr Expand the TransitinResults grup. Frm the expanded TransitinResults grup, add the fllwing fields: RetentinTime retentin time at the maximum intensity fr a transitin peak Fwhm full width at half max fr the transitin peak StartTime retentin time at the starting transitin peak integratin bundary EndTime retentin time at the ending transitin peak integratin bundary Area area under the curve (AUC), minus backgrund, fr the transitin peak Height maximum intensity fr the transitin peak UserSetPeak indicates whether r nt (TRUE/FALSE) integratin brders fr a transitin were manually adjusted Als, uncheck the Pivt Replicate Name checkbx, at the bttm f the frm. The reprt template shuld nw cntain a lt mre detail than the riginal Overview reprt templat, and the Edit Reprt frm shuld lk like: 14

15 T rerder sme fields in this new reprt template, d the fllwing: Select PrteinName in the clumn list n the right. Click the up arrw n the far right until the PrteinName field is at the tp f the list. Yu can achieve whatever rdering suits yu best, in this way, using the up and dwn arrw buttns n the far right, and yu can remve fields yu may have added by mistake with the red X buttn abve the arrw buttns. This tutrial, hwever, will cntinue withut making any further changes t the clumn list. T preview the values fr the current dcument in the reprt template yu have created, d the fllwing: Click n the Preview buttn. 15

16 When yu have reviewed the data, clse the preview frm. With data like this, where each peptide has bth a light analyte and a matching heavy istpe labeled internal standard, it may be mre cnvenient t wrk with the data fr these paired precursrs all in a single rw t allw yu t cmpare matching transitin values mre easily. T make this change t the Study 7 reprt template, d the fllwing: Check the Pivt Istpe Label check bx. Click n the Preview buttn again t see hw the reprt preview has changed. All f the values fr matching light and heavy transitins have been added t the same rw. New clumns have been added t achieve this, and clumns specific t an istpe label type have been given the prefixes light and heavy. The Preview Reprt frm is shwn belw, scrlled t shw the transitin between light and heavy clumn names: Yu might ntice that the clumn heavy IstpeLabelType shws heavy n every rw, and that there is als a clumn light IstpeLabelType which shws light n every clumn. When yu are pivting n a clumn, it des nt add much t include it in yur clumn list. Clse the Preview Reprt frm, and d the fllwing t remve the IstpeLabelType field frm this reprt: 16

17 Select IstpeLabelType in the clumn list n the right. Click the red X buttn n the far right edge f the clumn list. Yu can click the Preview buttn again t verify that bth the light IstpeLabelType and the heavy IstpeLabelType clumns have been remved. The next step in this tutrial, hwever, is t save yur wrk by ding the fllwing: Click the OK buttn in the Edit Reprt frm. Click the OK buttn in the Edit Reprts frm. This will return yu t the Exprt Reprt frm, which shuld nw lk like: Frm here, yu culd nw cntinue t exprt the new Study 7 reprt t a CSV file by selecting it and clicking the Exprt buttn, r click Share buttn t create file fr sharing this new reprt. Fr this tutrial, hwever, click the Cancel buttn t return t the Study7_example dcument. Quality Cntrl Summary Reprts S far yu have created reprts based n the values Skyline ffers fr each imprted replicate f the peptides and transitins specified in a dcument. Skyline als prvides reprt fields with summary statistics acrss all imprted replicates. These summary fields are particularly suited t quality cntrl by ensuring that replicate QC runs shw gd reprducibility and cefficients f variatin (CV). Skyline reprt templates can be designed with mean values and CV ver all replicates fr key parameters, such as peak area, FWHM and retentin time. 17

18 This tutrial prvides a summary reprt template that yu will imprt int Skyline, but it als describes hw t design such a summary reprt template in the Edit Reprt frm. T get started, first pen a new dcument by ding the fllwing: On the File menu, click Open. Navigate t the CustmReprts flder yu created fr this tutrial. (may nt be necessary) Select the Study9pilt.sky file. Click the Open buttn. This file cntains data acquired by ne CPTAC site ver 5 LC MRM MS runs, injecting 10 analyte peptides at a cnstant cncentratin f 50 fml ver all 5 runs. Such a dataset culd ptentially be used t assess reprducibility f replicate injectins. T generate a summary reprt fr key quality cntrl metrics fllw the steps belw: On the File menu, chse Exprt, and click Reprt. Click the Imprt buttn in the Exprt Reprt frm. Select the Summary_stats.skyr file frm the CustmReprts flder in the Open frm. Click the Open buttn. A new reprt template named Summary Statistics shuld have been added t the Reprt list, and the Exprt Reprt frm shuld nw lk like: T see the reprt fields included in the Summary Statistics reprt template, d the fllwing: Click the Edit List Buttn in the Exprt Reprt frm. 18

19 Select Summary Statistics in the Reprt list f the Edit Reprts frm. Click the Edit Buttn. Skyline displays the Edit Reprt frm with many mean and CV field names in the reprt clumn list n the right. T see where these fields came frm, perfrm the fllwing steps: Expand the field grup Peptides by clicking n the + icn t its left. Expand the field grup Precursrs. Expand the field grup PrecursrResultsSummary. The Exprt Reprt frm shuld nw lk like the image belw (withut the blue dts, which are added t indicate the fields already added t this reprt template): T preview this reprt n the data in the current dcument: Click the Preview buttn in the Edit Reprt frm. 19

20 The Preview Reprt frm is shwn lking like: This reprt shws that the data in Study9pilt.sky, taken frm 5 replicate injectins f the same sample, prduce peptide CV values fr FWHM mstly belw 5% and TtalArea in mst cases under 10% as indicated fr the crrespnding clumns with red bxes. In additin, the clumn called RangeBestRetentinTime (red bx) lists the RT drift in minutes fr any specific peptide ver the 5 replicate injectins. In this case there is nly minimal RT drift (< 0.15 min, < 10 sec). These summary reprts can be used t flag quality cntrl issues early, befre they impact data acquired n imprtant samples. In this particular dataset, HPLC and MS seem t behave acceptably. In ne case, hwever, fr the peptide INDISHTQSVSAK (frm prtein LEP), the CV value fr TtalArea is 59.2% and fr FWHM is 23.5% (circled in red), significantly differing frm the CV values f all the ther peptides. T return t the Skyline dcument and inspect this peptide mre clsely, d the fllwing: Clse the Preview Reprt by clicking n the red X in the upper right crner. Click the Cancel buttn in the Edit Reprt frm. Click the Cancel buttn in the Edit Reprts frm. Click the Cancel buttn in the Exprt Reprt frm. Select the peptide INDISHTQSVSAK in the peptide tree view. On the View menu, chse Peak Areas, and click Replicate Cmparisn (F7). 20

21 The Peak Areas view, shwing the ttal peak area and the cntributin f each transitin fr all 5 replicates, help clarify the prblem that was indicated in the CV values f the summary reprt preview. Fr cmparisn, select several f the ther peptides. The Peak Areas view will shw much better peak area reprducibility, as indicated by their much lwer TtalArea CV values in the summary reprt preview, where the majrity f the peptides had CV values < 10%. Skyline summary reprts can be used with quality cntrl data t flag issues early, while Skyline itself prvides pwerful visual displays t help get t the rt f the prblem quickly. Results Grid View In sme cases, yu may want t see sme f the values presented in reprts while yu are editing the data, fr immediate access t raw values displayed in the Skyline charts. The Skyline Results Grid view prvides real time access t many f the data fields available in the custm reprts. In the final sectins f this tutrial, yu will learn t use the Results Grid t gain immediate access t imprtant values and t anntate yur data t capture imprtant human insights, as yu inspect and refine yur mass spectrmeter utput in Skyline. T get started using the Result Grid view, d the fllwing: On the View menu, click Results Grid (Alt F2). Skyline shuld nw lk smething like this: 21

22 Nw rearrange the windws fr better viewing, by ding the fllwing: Click the Results Grid captin and drag it t the bttm edge f the main windw t dck it. Click the Peak Areas captin and drag it t the right edge f the main windw t dck it. Click and drag the splitters between the varius windw panes t adjust the space allcatin. On the View menu, chse Aut Zm, and click Best Peak, if it is nt already selected. Select the precursr element belw the currently selected peptide. This shuld leave Skyline lking smething like: 22

23 Skyline keeps its charts and the Results Grid in synch as yu navigate the data. If yu click n ne f the ther rws in the Results Grid, Skyline changes the active chrmatgram replicate tab. It als changes which bar in the Peak Areas graph has the selectin rectangle arund it, as well as the selectin in the Replicates list abve the peptide tree view. Nw click n a different chrmatgram chart tab, bar in the Peak Areas chart, r change the selectin in the Replicates list. The ther views will update, including the selected rw in the Results Grid. The first clumn t the right f the Replicate Name clumn in the Results Grid is the Precursr Replicate Nte. This clumn allws yu t assciate a free text nte with the PrcursrResults field grup fr the selected precursr and replicate. Try typing Lw signal in the first rw with replicate name Rep1 fr the currently selected peptide. All f the ther clumns in the current Results Grid display are calculated by Skyline and cannt be edited, but yu can change which clumns get displayed and in what rder. Several f the clumns are scrlled ut f view in the image abve. T reduce the number f precursr results clumns being displayed, d the fllwing: Right click n the clumn headings, and click Chse Clumns. Uncheck the clumns Min Start Time, Max End Time and Library Dt Prduct. Click the OK buttn. T adjust the clumn rder, d the fllwing: 23

24 Click the Ttal Area clumn header and drag it t the left edge f the Precursr Replicate Nte clumn. Skyline shuld nw lk smething like: Yu can nw review the results fr ther precursrs by ding the fllwing: Click in the peptide tree view. Use the up and dwn arrw keys t change the selectin t ther precursr elements. Finally expand a precursr and review its transitins. Yu culd apply the same technique t custmize the clumns being displayed fr transitins. Instead, cntinue nw t the final sectin in this tutrial t learn hw t add a custm anntatin that yu can cntrl with the Results Grid, and later exprt in a Skyline reprt. Custm Anntatins Skyline supprts three types f custm anntatins: 1. Free text (similar t the Nte which is available by default n all types) 2. True/False 3. Value list 24

25 The last tw types can be used t cllect infrmatin in a cntrlled vcabulary, as yu prcess yur data, which can later be exprted in a Skyline reprt and used as input fr prgrammatic statistical analysis. T define a simple new True/False anntatin, perfrm the fllwing steps: On the Settings menu, click Anntatins. Click the Edit List buttn n the Anntatin Settings frm. Click the Add buttn n the Define Anntatins frm. Type Tailing in the Name field n the Define Anntatin frm. Select True/False in the Type field. Check the Precursr Results checkbx in the Applies T list. The Define Anntatin frm shuld nw lk like: Nw finish defining the anntatin and add it t yur dcument by ding the fllwing: Click the OK buttn n the Define Anntatin frm. Click the OK buttn n the Define Anntatins frm. Check the checkbx fr the new Tailing anntatin. The Anntatin Settings frm shuld nw lk like: 25

26 Click the OK buttn. Select the secnd t last precursr element fr the peptide ESDTSYVSLK. Yu shuld see the new Tailing clumn added as the last clumn in the Results Grid. Click n it and drag it t the left edge f the Precursr Replicate Nte clumn where it can be mre easily edited. Yu can als click and drag the lines between the clumn headers t resize the clumns fr easy viewing and editing. The Skyline windw shuld nw lk smething like: 26

27 The peaks in this dcument d nt have a lt f issues, but this ne des have sme peak tailing. Check the checkbx in the new Tailing clumn fr the Rep1 rw. Change selectin t each f the ther 5 rws. Decide whether yu think the tailing is wrth nting and check the checkbx, if yu think it is. Change the selected item in the precursr tree and then return t the precursr t verify that yur changes were recrded. Yu can nw exprt the new Tailing anntatin in a Skyline custm reprt. It will appear in the PrecursrResults field grup. Cnclusin This tutrial has intrduced yu t the flexible custm reprts Skyline ffers fr accessing the rich set f values assciated with yur Skyline targeted prtemics dcuments. These reprts ffer a smth path frm data analysis within Skyline t mre cmplex statistical analysis with Excel r custm prgrams in R, Matlab, Java r C++. Yu have learned abut summary statistics available thrugh these reprt templates, which can prvide quick indicatrs f data quality. Yu can share yur custm reprt templates with cllabratrs r as supplementary material in yur publicatins, helping thers t repeat yur analyses with new data sets even frm different instrumentatin, taking full advantage f the Skyline multi vendr supprt. Finally, yu learned hw t use the Skyline Results Grid t gain immediate access t many f the values available in Skyline reprts and t wrk with custm anntatins t add rich new infrmatin t yur dcuments. If yu were nt aware f these features befre, they will certainly increase the scpe f experiment yu can achieve with Skyline. 27

28 Glssary f Custm Reprt Fields Prteins (tp level): PrteinName Prtein Name as defined r entered int Skyline (in sme cases in cnjunctin with the accessin number). PrteinDescriptin When prteins are imprted int Skyline thrugh public FASTA sequence files, a Prtein Descriptin is prvided. Yu can see the Prtein Descriptin als when yu hver ver the prtein name in the peptide tree view. PrteinSequence When prteins are imprted int Skyline thrugh public FASTA sequence files the full prtein sequence is accessible. Yu can see the prtein sequence als when yu hver ver the prtein name in the peptide tree view. PrteinNte A free text nte assciated with the prtein by clicking Edit Nte n the Edit menu. Results (tp level): ReplicateName The replicate name assigned t the data during imprt. ReplicatePath Reserved fr future use. FileName The name f the file frm which the data was imprted. SampleName The sample name, if the data was imprted frm a multi sample WIFF file, r the file name again fr ther file types. MdifiedTime Last time and date at which the riginal mass spectrmeter file was mdified n disk. AcquiredTime Last time and date at which the mass spectrmeter began acquiring this replicate data. Or #N/A if the file was imprted with a versin lder than 1.1. Peptides: PeptideSequence Amin acid sequence f the peptide BeginPs The psitin f the first (N terminal) amin acid f the peptide within its cntaining prtein sequence, r #N/A if n prtein sequence is available. EndPs The psitin f the last (C terminal) amin acid f the peptide within its cntaining prtein sequence, r #N/A if n prtein sequence is available. PredictedRetentinTime The retentin time predicted by a retentin time regressin between a set f measured results and a retentin time calculatr (currently nly SSRCalc 3.0 is available), r #N/A if n retentin time regressin has been assigned. AverageMeasuredRetentinTime The average peptide retentin time ver all replicates. PeptideNte A free text nte assciated with the peptide by clicking Edit Nte n the Edit menu. PeptideResults (per replicate values): 28

29 PeptidePeakFundRati The rati f transitins fr which a peak was measured t the ttal number f transitins in the peptide. Peak indicatrs in the peptide tree view crrespnd t green = 1.0 (all transitins integrated), range >= 0.5, red < 0.5. PeptideRetentinTime Peptide retentin time fr each replicate run. RatiTStandard Peptide area rati f light t heavy. Precursrs: Charge/PrecursrCharge The charge assciated with the precursr in. IstpeLabelType A label type name assciated with the precursr in (light/heavy), indicating which istpe mdificatins are applied. PrecursrNeutralMass Neutral mass f the precursr in Daltns. Mz/PrecursrMz The mass t charge rati (m/z) f the precursr in. CllisinEnergy Cllisin Energy fr the precursr in accrding t instrument/vendr specific default cllisin energy equatin within Skyline. DeclusteringPtential Declustering Ptential fr the precursr in accrding t instrument/vendr specific default declustering ptential equatin within Skyline. MdifiedSequence Peptide sequence including any amin acid mdificatins such as cysteine alkylatin. (example: AGLC[+57]QTFVYGGC[+57]R) PrecursrNte A free text nte assciated with a precursr by clicking Edit Nte n the Edit menu. LibraryName The name f a MS/MS spectral library, if a MS/MS spectral library spectrum is assciated with the precursr in. LibraryType The type f MS/MS spectral library (BibliSpec, GPM, NIST), if a MS/MS spectral library spectrum is assciated with the precursr in. PrecursrLibraryRank A MS/MS spectral library ranking f the precursr amng ther precursrs fr a prtein that match supplied spectral libraries, using a scre specified in the peptide library settings. LibraryScre1 Raw peptide library scre that may r may nt be used t rank amng precursrs f a prtein. LibraryScre2 Raw peptide library scre that may r may nt be used t rank amng precursrs f a prtein. LibraryScre3 Raw peptide library scre that may r may nt be used t rank amng precursrs f a prtein. PrecursrResults (per replicate values): PrecursrPeakFundRati The rati f transitins fr which a peak was measured t the ttal number f transitins in the precursr. Peak indicatrs in the peptide tree view crrespnd t green = 1.0 (all transitins integrated), range >= 0.5, red < 0.5. BestRetentinTime The RetentinTime value f the transitin with the highest maximum intensity fr the precursr. MaxFwhm The maximum full width at half max (FWHM) f the transitins fr the precursr. 29

30 MinStartTime Minimum StartTime f all transitins fr a precursr. Unless manually edited all transitins fr a precursr use the same integratin bundaries. MaxEndTime Maximum EndTime f all transitins fr a precursr. Unless manually edited all transitins fr a precursr use the same integratin bundaries. TtalArea The summed Area values f all individual transitins fr the particular precursr. TtalBackgrund The summed Backgrund values f all individual transitins fr the particular precursr. TtalAreaRati The rati f the TtalArea f this precursr t the first internal standard label type, befre versin 0.7 this was always light/heavy, and appeared n the heavy precursr. TtalAreaNrmalized The TtalArea nrmalized t the sum f the TtalArea values fr all peptide precursrs in the dcument. CuntTruncated The number f transitins fr a precursr that integrate a peak with a bundary at either end f the acquisitin time range, where intensity at the end is greater than 1% f the entire peak height higher than the ther extent. Identified True if a MS/MS peptide identificatin exists fr the result file at a time between the peak integratin bundaries. (new in v1.2) LibraryDtPrduct The dt prduct (described in the riginal BibliSpec article between the individual transitin peak areas f the precursr and the intensities f the matching in peaks in the matched MS/MS spectral library spectrum, r #N/A if the precursr has nt matching library spectrum r has fewer than 4 transitins. This is a useful value fr methd refinement. It wrks best when 6 r mre transitins are present. IstpeDtPrduct The dt prduct calculatin described abve, but between the individual precursr (M, M+1, M+2, etc.) peak areas f the precursr and the intensities f the predicted istpe distributin, r #N/A if the transitin is nt a precursr istpe, r the chrmatgram was nt extracted frm high reslutin MS1 data. (new in v1.2) UserSetTtal True if the default chice f peak r its bundaries was manually altered. OptStep Optimizatin step value indicating distance frm the default value fr the parameter being ptimized, 0 fr the default parameter value, r if n ptimizatin is being perfrmed in the replicate. OptCllisinEnergy The cllisin energy value crrespnding t the OptStep if cllisin energy ptimizatin is being perfrmed. OptDeclusteringPtential The dlecustering ptential value crrespnding t the OptStep if declustering ptential ptimizatin is being perfrmed. PrecursrReplicateNte A free text nte assciated with a result set f the precursr using the Results Grid. PrecursrResultsSummary (summary statistics acrss all replicates): MinBestRetentinTime Minimum f the precursr BestRetentinTime values. MaxBestRetentinTime Maximum f the precursr BestRetentinTime values. 30

31 RangeBestRetentinTime The difference between MaxBestRetentinTime and MinBestRetentinTime, which can be used t gauge the spread f retentin times measured. MeanBestRetentinTime Mean f the precursr BestRetentinTime values. StdevBestRetentinTime Standard deviatin f the precursr BestRetentinTime values. CvBestRetentinTime Cefficient f variatin (CV) f the precursr BestRetentinTime values. MeanMaxFwhm Mean f the precursr MaxFwhm (peak width) values. StdevMaxFwhm Standard deviatin f the precursr MaxFwhm (peak width) values. CvMaxFwhm Cefficient f variatin (CV) f the precursr MaxFwhm (peak width) values. MeanTtalArea Mean f the precursr TtalArea values. StdevTtalArea Standard deviatin f the precursr TtalArea values. CvTtalArea Cefficient f variatin (CV) f the precursr TtalArea values. MeanTtalAreaRati Mean f the precursr TtalAreaRati values. StdevTtalAreaRati Standard deviatin f the precursr TtalAreaRati values. CvTtalAreaRati Cefficient f variatin (CV) f the precursr TtalAreaRati values. MeanTtalAreaNrmalized Mean f the precursr TtalAreaNrmalized values. StdevTtalAreaNrmalized Standard deviatin f the precursr TtalAreaNrmalized values. CvTtalAreaNrmalized Cefficient f variatin (CV) f the precursr TtalAreaNrmalized values. Transitins: PrductCharge Charge (z) f the prduct in. PrductNeutralMass Neutral mass f the prduct in peptide fragment in Daltns. PrductMz The mass t charge rati (m/z) f the prduct in. FragmentIn The name f the prduct in peptide fragment (e.g. y8, y10, b7, etc.). FragmentInType The type f the prduct in (y, b, c, z, a, x, precursr) FragmentInOrdinal Psitin f the amin acid in the peptide after (C terminal f) which the peptide was cleaved upn fragmentatin. (e.g. 8, 10, 7, etc.) CleavageAa Specific amin acid residue in the peptide after (C terminal f) which the peptide was cleaved upn fragmentatin. (e.g. P, M, S, T, etc.) TransitinNte A free text nte assciated with the transitin by clicking Edit Nte n the Edit menu. LibraryRank Intensity ranking f the MS/MS spectral library fragment in peak matching the prduct m/z fr this transitin, if a matching spectrum is assciated with the precursr. LibraryIntensity Raw Intensity value f the MS/MS spectral library fragment in peak matching the prduct m/z fr this transitin, if a matching spectrum is assciated with the precursr. 31

32 TransitinResults: RetentinTime Retentin time at the maximum intensity fr a transitin peak. Fwhm Full width at half max (FWHM) fr the transitin peak. FwhmDegenerate True if ne f the peak integratin bundaries intersects the chrmatgram abve the half maximum intensity. StartTime Retentin time at the starting integratin bundary fr the transitin peak. EndTime Retentin time at the ending integratin bundary fr the transitin peak. Area Area under the curve (AUC), minus backgrund, fr the transitin peak. Backgrund The area f the rectangle frmed by the integratin bundaries, and the baseline and a line perpendicular t minimum intersectin intensity between the integratin bundaries and the chrmatgram fr the transitin peak. AreaRati The rati f the Area f this transitin t its crrespnding transitin in the first internal standard label type, befre versin 0.7 this was always light/heavy, and appeared n the heavy transitins. AreaNrmalized The Area nrmalized t the sum f the Area values fr all transitins in the dcument. Height The maximum intensity f the pints between the transitin peak integratin bundaries. Truncated True if the integrated a peak has a bundary at either end f the acquisitin time range, where intensity at the end is greater than 1% f the entire peak height higher than the ther extent. Or #N/A if the data was imprted with a versin lder than 1.1. PeakRank The rank based n Area f this transitin amng all ther transitins f the same precursr. UserSetPeak True if the default chice f peak r its bundaries was manually altered. TransitinReplicateNte A free text nte assciated with a result set f the transitin using the Results Grid. LibraryRank The rank based n LibraryIntensity f this transitin amng all transitins allwed by the transitin Filter settings, shwn in the user interface as (rank #). LibraryIntensity The MS/MS peak intensity crrespnding t the transitin prduct in in the matching library spectrum. IstpeDistIndex Zer fr the mnistpic peak, 1 fr M+1, 2 fr M+2, etc. (new in v1.2) IstpeDistRank The rank based n the IstpeDistPrprtin amng all istpe peaks fr the predicted istpe distributin, shwn in the user interface as (irank #). Currently nly available fr precursr transitins filtered frm high reslutin MS1 scans. (new in v1.2) IstpeDistPrprtin The prprtin f the entire istpe distributin predicted fr this istpe peak. Currently nly available fr precursr transitins filtered frm high reslutin MS1 scans. (new in v1.2) 32

33 TransitinResultsSummary (summary statistics acrss all replicates): MinRetentinTime Minimum f the transitin RetentinTime values. MaxRetentinTime Maximum f the transitin RetentinTime values. RangeRetentinTime The difference between MaxRetentinTime and MinRetentinTime, which can be used t gauge the spread f retentin times measured. MeanRetentinTime Mean f the transitin RetentinTime values. StdevRetentinTime Standard deviatin f the transitin RetentinTime values. CvRetentinTime Cefficient f variatin (CV) f the transitin RetentinTime values. MeanFwhm Mean f the transitin Fwhm (peak width) values. StdevFwhm Standard deviatin f the transitin Fwhm (peak width) values. CvFwhm Cefficient f variatin (CV) f the transitin Fwhm (peak width) values. MeanArea Mean f the transitin Area values. StdevArea Standard deviatin f the transitin Area values. CvArea Cefficient f variatin (CV) f the transitin Area values. MeanAreaRati Mean f the transitin AreaRati values. StdevAreaRati Standard deviatin f the transitin AreaRati values. CvAreaRati Cefficient f variatin (CV) f the transitin AreaRati values. MeanAreaNrmalized Mean f the transitin AreaNrmalized values. StdevAreaNrmalized Standard deviatin f the transitin AreaNrmalized values. CvAreaNrmalized Cefficient f variatin (CV) f the transitin AreaNrmalized values. 33