proteomics workflows share one essential 
 requirement : they necessitate appropriate quality control ( qc ) .
stringent 
 qc not only requires an evaluation of outcomes , rather qc starts with 
 adequate experimental design ; it incorporates monitoring of processes , 
 protocols , and reagents ; and it includes aspects such as adequate 
 training of personnel and a surveillance of the proper functioning 
 of instrumentation . for proteomics workflows , qc must be established 
 at four steps : sample preparation , liquid chromatography ( lc ) , mass 
 spectrometry ( ms ) , and data interpretation .
a recent study showed that a major reason for the failure of a number 
 of laboratories to reliably identify proteins present in a relatively 
 simple test sample was due to inadequate data processing ; the same 
 study suggested that this could be overcome by standardized operating 
 procedures involving appropriate protocols for data interpretation .
this is reassuring as a bioinformatics data interpretation 
 workflow , once established , can be maintained easily because software 
 algorithms continue to work reproducibly with moderate requirement 
 for maintenance .
in contrast , all the other above - mentioned steps 
 are subject to variation from experiment to experiment , and suboptimal 
 system performance needs to be identified and corrected promptly . 
 for this reason , software which facilitates an effortless monitoring 
 of the performance of lc and ms systems
improvements in lc and ms instrumentation contribute to the remarkable 
 performance of today s lc  ms equipment , empowering researchers 
 to implement novel experimental protocols .
however , challenging applications 
 require measures to ensure constant and optimal system performance . 

highly sophisticated qc approaches were reported previously such as 
 the nist msqc pipeline , which outputs 46 qc metrics , or quameter a software which can compute up to 42 qc metrics 
 for instruments from different vendors .
qc software has also been reported for the detection of lc runs 
 that constitute outliers and for computation 
 and visualization of qc metrics from individual lc runs , and a real - time qc solution for monitoring electrospray 
 quality and lc backpressure was described recently . however , to our knowledge no currently available qc software 
 allows an automated upload of qc runs to a dedicated server postacquisition , 
 followed by seamless computation of qc metrics based on peptide identifications 
 and instrument parameters , storage of qc results in a database , and 
 the possibility for convenient plotting of the time course of qc metrics 
 via a web browser .
such a longitudinal analysis of qc data from multiple 
 lc runs could potentially reveal suboptimal lc 
ms system performance 
 as a deviation outside the range of a qc metric when the lc 
we therefore developed 
 simpatiqco ( simple automatic quality control ) , a software suite specifically designed to aid routine longitudinal 
 monitoring of qc metrics and troubleshooting when using ltq orbitrap , 
 q - exactive , ltq ft , and ltq instruments .
simpatiqco is a server - based 
 application , designed to process raw files from the above - mentioned 
 thermo scientific instruments that can be uploaded either manually 
 ( individually or in batch ) via a web browser interface or in an automated 
 manner via a  hot folder 
( windows network share or 
 ftp ) . as xcalibur can be configured to run a batch file postacquisition , 
 raw files can be copied instantaneously from mass spectrometers in 
 the laboratory to the  hot folder  on the simpatiqco 
 host for automatic processing .
simpatiqco then extracts information 
 from the raw file , starts a mascot search to identify spectra , and 
 calculates qc metrics .
results are stored in a database and can be 
 viewed for individual qc runs as well as plotted over time via a web 
 browser interface from any computer in the laboratory . to demonstrate 
 the successful usage and general utility of the software suite , we 
 present longitudinal qc data from six instruments and four laboratories 
 in the supporting information ( figure s-1 ) . 

three of the four laboratories installed a simpatiqco server in - house , 
 while one laboratory uploaded data to a simpatiqco server installed 
 and operated by one of the other laboratories ( which is an option 
 for laboratories that do not have the resources to maintain the server 
 themselves ) .
we also propose an lc  ms quality control 
 workflow based on the routine surveillance of qc metrics with two 
 types of qc samples that are intercalated with regular samples to 
 monitor two disparate aspects of overall system performance : a sample 
 of very low complexity designed to monitor sensitivity using an analyte 
 ( peptide ) concentration only modestly above the limit of detection 
 ( lod ) of a system performing close to optimum ; and another sample 
 of very high complexity that poses a challenge to the speed of data 
 acquisition .
metrics derived from these 
 samples thus reflect the sensitivity as well as the speed and overall 
 performance of the lc  ms system . of note
, simpatiqco can be 
 configured to accommodate other types of samples , as disparate qc 
 samples are in use within the community . indeed , some of the above - mentioned 
 participating laboratories preferred to use different standard proteins 
 and synthetic peptides and different concentrations in their qc samples .
the purpose of simpatiqco is to provide a set of key qc metrics 
 that are generated in an effortless manner and that can be displayed 
 conveniently .
simpatiqco facilitates a longitudinal surveillance of 
 the time course of system performance and within - laboratory reproducibility , 
 and it helps pinpoint potential suboptimal system performance .
identification 
 of outliers with regard to the performance metrics should prompt an 
 immediate enquiry so that the reason for deviant performance can be 
 identified and resolved . in this way
, simpatiqco may aid proteomics 
 laboratories achieve and maintain optimum quality of data acquisition .
raw files from qc runs intercalated with regular 
 samples are uploaded to a server .
simpatiqco extracts data such as 
 ion injection times , tic , and lock mass information , and estimates 
 the overfill or underfill ratio of the automatic gain control ( agc ) 
 target value for each spectrum .
ms / ms 
 spectra are submitted to a mascot server for peptide identification . 

simpatiqco then calculates qc metrics that reflect the performance 
 of lc , electrospray , and ms such as peak widths and areas , peptide 
 elution times and intensities , tic , the number of identified peptide 
proteins 
 of interest  . for each qc metric the range indicating optimal 
 system performance
is learned from the data and displayed using a 
 background band colored in green , whereas yellow or red colored bands 
 highlight deviant system performance .
we calculate robust statistics 
 on qc metrics , eliminating the requirement to manually remove outliers 
 (  bad runs  ) .
php scripts and apache webserver allow comfortable display of results 
 from any computer in the laboratory .
a description of the qc 
 samples and the lc and ms methods used by the four laboratories is 
 provided in the supporting information , 
 illustrating that simpatiqco is compatible with different qc work 
 flows established in various laboratories .
a detailed overview of 
 the components and design of the simpatiqco software package is also 
 provided in the supplementary materials and methods section within 
 the supporting information .
further information , 
 download links , installation guideline , manual , and faqs are available 
 online at http://ms.imp.ac.at/.
obtaining mass spectrometry 
 data for state - of - the - art proteomics studies requires qc measures . 
 in our view ,
the key to achieve and maintain optimum data quality 
 is successive , routine , and if possible automated rather than sporadic 
 and manual monitoring of system performance . in the following we present 
 a qc workflow as well as a software package aimed to aid the maintenance 
 of constant and optimum lc  ms system performance .
traditionally 
 the lc has been a major source of concern , and close surveillance 
 of lc performance is certainly an important prerequisite to ensure 
 high quality proteomics data .
however , in our experience there is 
 likewise a requirement to monitor the performance of the ms instrumentation 
 as closely as that of the lc .
for instance with the newer generations 
 of orbitrap instruments that have enhanced sensitivity partly due 
 to increasing the overall gas throughput into the system , there may be the need to clean various components 
 of the instrument more often in order to maintain optimum performance , 
 as deposition of particles onto components of the ion optics can lead 
 to a decrease in ion transmission .
both lc and ms instruments 
 offer a variety of instrument - specific system tests .
for instance , 
 the latest version of velos orbitrap instrument software ( tune 2.7 
 sp1 ) offers several diagnostic procedures that have been added to 
 assist in determining whether a deposition of charged particles is 
 occurring and which part of the ion optics most likely has the contamination
. 
 however , several types of system evaluation procedures on orbitrap 
 instruments may require switching from the nanosource to the esi source , 
 interrupting measurements in addition to the time required for the 
 tests themselves .
for this reason system tests are costly to perform 
 on a daily or more than daily basis .
in addition there is a risk that 
 the mounting and unmounting of system parts to perform these tests 
 might lead to a deterioration of system performance aside from the 
 time requirements .
cleaning procedures and the associated bake - out 
 of the instrument lead to prolonged instrument downtime , so that there 
 is a requirement to estimate appropriate timing of such maintenance 
 procedures .
we therefore reasoned that it would be desirable to have 
 a system evaluation test at hand that can be performed without any 
 need to interrupt the running lc  ms system .
we hence 
 suggest that the more time - consuming instrument - specific tests such 
 as those mentioned above be performed only at defined time points 
 dedicated to system maintenance and during setup or troubleshooting 
 of an lc  ms system . in these situations , considerable time 
 and effort is invested into optimizing system performance .
manual 
 evaluation of raw files and analysis with tools such as rawmeat ( http://vastsci.com/rawmeat/ ) , nist msqc , quameter , logviewer , and maxquant can 
 provide valuable information at this stage .
in addition parameters 
 for methods should be tested in order to evaluate their effect on 
 system performance .
these time - consuming optimization steps are necessary 
 because monitoring the time course of performance metrics can only 
 be regarded as an indicator of optimum system performance when the 
 values of the metrics under optimal conditions have been established 
 in the first place .
after these initial steps , we suggest a routine 
 analysis of two types of qc samples in short time intervals to monitor 
 qc metrics such as sensitivity as well as speed and overall performance 
 of the entire lc  ms system .
the qc samples are measured intercalated 
 within the regular experiments , and analyzed automatically with the 
 help of simpatiqco so that there is no interruption of system operation .
the general concept is based on the consideration that relevant 
 system alterations involving a significant change in system performance 
 should lead to a change of qc metrics derived from either or both 
 of the two qc samples . as we want the qc samples to indicate whether 
 the lc 
ms system is in an adequate status for measuring actual 
 samples , the choice of the two qc samples is such that one qc sample 
 is designed to evaluate sensitivity ( qc 1 ) whereas the other one is 
 intended to monitor overall system performance and speed ( qc 2 ) .
the 
 choice of a suitable amount of the qc 1 sensitivity sample is important . 

one may first perform a dilution series to establish the lod and then 
 decide to inject an amount only modestly above this value .
when the 
 injected amount of qc 1 sample is too high , the test would constitute 
 a less sensitive indicator of impaired system performance .
however 
 a concentration that is too low would also be suboptimal because this 
 would lead to on / off observations rather than a more robust performance 
 required for the calculation of meaningful performance statistics 
 on the qc metrics .
specifically the two qc samples that we suggest 
 are as follows:1.a sensitivity analysis , typically based 
 on measuring a small amount of a well - defined sample such as , e.g. , 
 1 fmol of bsa , 2.5 fmol of a mixture of 10 phosphopeptides , and 500 
 fmol of cytochrome c over a short , e.g. , a 0.5 h 
 gradient time ( see supplementary materials and methods section of 
 the supporting information ) .
sample whenever there have been 
 alterations to any part of the lc  ms system , after calibration 
 of the ms ( change to esi source ) , and in general at least once or 
 if possible several times per day.2.a performance and speed analysis , usually based on the 
 injection of a highly complex sample such as 0.1 g or 1 g 
 of hela measured over a longer gradient time ( for instance 3 h ) .
this 
  qc 2  sample is measured at least once per week , whenever 
 there have been alterations to any component of the lc  ms system , 
 and before the analysis of biological samples that require high speed 
 of acquisition and optimal overall performance . a sensitivity analysis , typically based 
 on measuring a small amount of a well - defined sample such as , e.g. , 
 1 fmol of bsa , 2.5 fmol of a mixture of 10 phosphopeptides , and 500 
 fmol of cytochrome c over a short , e.g. , a 0.5 h 
 gradient time ( see supplementary materials and methods section of 
 the supporting information ) .
sample whenever there have been 
 alterations to any part of the lc  ms system , after calibration 
 of the ms ( change to esi source ) , and in general at least once or 
 if possible several times per day . a performance and speed analysis , usually based on the 
 injection of a highly complex sample such as 0.1 g or 1 g 
 of hela measured over a longer gradient time ( for instance 3 h ) .
this 
  qc 2  sample is measured at least once per week , whenever 
 there have been alterations to any component of the lc  ms system , 
 and before the analysis of biological samples that require high speed 
 of acquisition and optimal overall performance .
other types of qc samples such as , e.g. , other proteins , 
 synthetic peptides spiked at different concentrations , escherichia 
 coli , pyrococcus furiosus , or yeast , are 
 conceivable and can be configured for automatic analysis with simpatiqco . 
 a schematic overview of the simpatiqco software suite is shown in 
 figure 1 .
two types of qc samples are analyzed on each lc  ms 
 system : qc 1 contains a simple digested protein mixture ( e.g. , cytochrome c , bsa , and synthetic phosphopeptides ) that is measured 
 12/day for an evaluation of sensitivity and lc metrics 
 such as peptide retention times .
qc 2 is a hela digest that is analyzed 
 12/week to evaluate system speed and overall performance . 

after upload of the respective raw files to the simpatiqco server , 
 qc metrics are calculated and stored in a postgresql database .
the 
 database is linked to a webserver and can be queried to visualize 
 the time course of qc metrics via a web browser .
a single simpatiqco server can be used to upload , 
 analyze , and visualize data from several lc  ms systems ( figure 1 and figure s-2 in the supporting 
 information ) .
allows access to analyses of individual 
 uploaded raw files , and manual upload and deletion of raw files .
 time 
 course  permits visualization of the time course of qc metrics 
 based on information stored in the database .
a virtual logbook can 
 be accessed under  log  where the date and a short description 
 of events such as system maintenance or cleaning of the ion optics 
 can be entered for each system . 
settings  can be used 
 to set the url of the mascot server used for searching ms / ms spectra 
 and to configure predefined 
run - types  and settings 
 for the calculation of statistics from performance metrics .  peptide 
 lists  allow a definition of peptides whose peak elution time , 
 apex intensity , peak width , and area can be plotted via the  time 
 course  menu .
 proteins of interest  define 
 proteins for which the sequence coverage is shown when clicking on 
 a raw file in the  raw files  menu , and for which the 
 time course of sequence coverage can be plotted in the 
 run - types  define settings specific 
 for a certain instrument and qc sample , such as precursor and fragment 
 ion tolerance , mascot  instrument type 
( esi trap , etd 
 trap , etc . ) , the fasta database , enzyme , and mascot peptide ion score 
 cutoff .
in addition the  settings  menu allows defining 
 the time period for the calculation of statistics on qc metrics ( default 
 setting : inclusion of all runs ) , and whether the green , yellow , and 
 red background bands should be calculated based on a certain multiple 
 ( e.g. , 1 , 2 , or 3 ) of the standard deviation or 
 the median absolute deviation ( mad ) .
after data upload , simpatiqco 
 derives several qc metrics from the raw files , searches ms / ms spectra 
 via mascot , and then stores all extracted data and results in a database 
 that can be queried remotely by a web browser client .
this is advantageous 
 because it provides researchers and technical personnel a convenient 
 access to the qc data reflecting the current performance of each lc  ms 
 system in the laboratory .
in addition , simpatiqco allows operators 
 to enter  system maintenance & service messages  
 into a virtual logbook , for instance when the s - lens was cleaned or 
 when a service engineer performed system maintenance work on the instrument . 

the respective messages can be superimposed on the time course plots 
 so that the effect of system maintenance can be related to changes 
 in the qc metrics ( figure 2 ) . in this way
, 
 simpatiqco can help identify impaired system performance , monitor 
 whether system maintenance procedures were successful , and thus help 
 achieve and maintain close to optimum lc  ms system performance 
 to ensure high quality data acquisition .
example of simpatiqco 
 output : bsa sequence coverage ( a ) and average ms ion injection 
 times ( b ) on a velos orbitrap system monitored over time .
system maintenance 
 and service messages were entered manually by an operator and illustrate 
 the relationship between the two qc metrics and system maintenance 
 efforts such as venting and cleaning of the s - lens and exit lens ( log 
 entry  s - lens cleaned  ) by laboratory staff ( which improved 
 bsa sequence coverage but led to an only transient reduction in average 
 ms injection times ) or venting and cleaning of the transfer 
 lens between the high pressure and the low pressure cells by a service 
 engineer .
, simpatiqco learns the range 
 reflecting adequate or inadequate system performance from the uploaded 
 data .
ms system was operated under 
 adequate or possibly optimum conditions for the majority of qc runs . 
 via the simpatiqco  raw files 
. however it may be difficult to judge 
 which data points actually represent outliers .
we therefore decided 
 to implement robust statistics to find the range for each metric that 
 indicates adequate system performance , calculating median and mad 
 ( median absolute deviation ) values as an alternative to average values 
 and standard deviation .
contrary to average values and standard deviations 
 that are sensitive to outliers , median values and mad provide a more 
 robust estimation of adequate system performance that is insensitive 
 to outliers .
the default setting is that these statistics are calculated 
 for all lc runs of a certain run type that have been uploaded to the 
 database .
however as soon as a time period that likely reflects  optimum 
 system performance  has been identified , operators may define 
 and configure this period in the  settings  menu as 
 a fixed reference time for calculation of statistics of qc metrics 
 such as median , mad , average values , and standard deviation . in this 
 way
, the above - mentioned statistics reflect the ranges during  optimum 
 system performance  . for longitudinal monitoring of the time 
 course of qc metrics , the values of the respective metric are plotted 
 against a colored background where a green color band indicates optimum 
 system performance ( median  mad ) whereas deviant system performance 
 is highlighted by yellow ( between 1 and 2  mad ) or red color 
 ( outside 2  mad ) .
these plots of the time course of qc metrics 
 allow convenient and rapid pinpointing of adequate or likely suboptimal 
 system performance .
simpatico automatically calculates and displays 
 the following qc metrics:1.metrics for which the time course can 
 be visualized ( figure 2 , figures s-1 and s-4 
 in the supporting information)number of ms and 
 ms / ms scansms and ms / ms scans where lock - mass was detectedms and ms / ms scans 
 where maximum injection time was reachedaverage ms lock mass deviationaverage ms and ms / ms ion injection time and total scan timetotal number of identified psms 
 and proteinssequence 
 coverage of proteins that match an entry in the  proteins of 
 interest  listfor definable  peptide lists 
( e.g. , for qc 1 sample : 
 cytochtome c peptides and synthetic phosphopeptides ) : 
 peptide peak elution time , apex intensity , peak width and area2.metrics that 
 can be plotted over the retention time of individual raw files ( figure 3)ms and ms / ms ion 
 injection time and scan timelock mass detection and deviationms and ms / ms ticms and ms / ms  ( % ) 
 of target valuenumber of psms identified per minutem / z of 
 triggered precursors metrics for which the time course can 
 be visualized ( figure 2 , figures s-1 and s-4 
 in the supporting information)number of ms and 
 ms / ms scansms and ms / ms scans where lock - mass was detectedms and ms / ms scans 
 where maximum injection time was reachedaverage ms lock mass deviationaverage ms and ms / ms ion injection time and total scan timetotal number of identified psms 
 and proteinssequence 
 coverage of proteins that match an entry in the  proteins of 
 interest  listfor definable  peptide lists  ( e.g. , for qc 1 sample : 
 cytochtome c peptides and synthetic phosphopeptides ) : 
 peptide peak elution time , apex intensity , peak width and area number of ms and 
 ms / ms scans ms and ms / ms scans where lock - mass was detected ms and ms / ms scans 
 where maximum injection time was reached average ms lock mass deviation average ms and ms / ms ion injection time and total scan time total number of identified psms 
 and proteins sequence 
 coverage of proteins that match an entry in the  proteins of 
 interest  list for definable  peptide lists 
( e.g. , for qc 1 sample : 
 cytochtome c peptides and synthetic phosphopeptides ) : 
 peptide peak elution time , apex intensity , peak width and area metrics that 
 can be plotted over the retention time of individual raw files ( figure 3)ms and ms / ms ion 
 injection time and scan timelock mass detection and deviationms and ms / ms ticms and ms / ms  ( % ) 
 of target valuenumber of psms identified per minutem / z of 
 triggered precursors ms and ms / ms ion 
 injection time and scan time lock mass detection and deviation ms and ms / ms  ( % ) 
 of target value  number of psms identified per minute m / z of 
 triggered precursors the above - mentioned metrics are calculated and can be 
 visualized for both types of qc runs .
however , we define peptide lists 
 only for the  sensitivity  qc 1 sample because the calculation 
 of retention times is more precise for this sample . taken together , 
 these qc metrics provide a quick overview and help in monitoring the 
 entire lc  ms system , including performance of the lc , electrospray , 
 and ms .
lc performance can be evaluated 
 via the peak apex retention times and intensities , peak widths , and 
 peak areas of peptides on definable  peptide lists  
 ( figure s-1 in the supporting information ) .
these metrics are mandatory to monitor particularly when reproducible 
 retention time and constant ionization efficiency are required , e.g. , 
 for label - free quantification .
there are myriads of reasons 
 for shifts in retention times such as alterations in solvent solutions , 
 temperature shifts , dead volume , and leaking connections . in general 
 malfunctioning of almost every part of the lc may lead to a change 
 in retention times ; for this reason this metric is a valuable measure 
 of lc performance .
retention time shifts may also indicate that the 
 lc fails to generate an adequate flow rate , e.g. , because of partial 
 occlusion of the trap column or the analytical column that the system 
 can not compensate .
in these cases , a change in the pressure curves 
 on the lc can confirm the cause .
the large amount of cytochrome c ( 500 fmol ) in the qc 1 sample permits a rapid inspection 
 of cytochrome c peptide retention times with lc software 
 whenever a uv detector is used .
the  peptide list  feature 
 of simpatiqco allows monitoring of this metric even without a uv detector 
 directly from mass spectrometry data .
additional usage of a uv detector 
 may still be helpful , e.g. , for the identification of peak broadening 
 due to dead volume in fluidic connections between the uv cell and 
 the electrospray emitter . moreover 
permit 
 monitoring the retention time , e.g. , of the phosphopeptides in the 
 qc 1 sample whose concentration would be too low to be detectable 
 on the uv trace .
in addition , peak widths can be plotted for 
 peptides on a simpatiqco  peptide list  .
again there 
 are many causes for peak broadening such as the presence of post - column 
 dead volume or a deterioration of the analytical column . in the absence 
 of a uv detector , simpatiqco  peptide lists 
provide 
 a possibility to monitor peak widths , which are an excellent indicator 
 of lc system performance .
monitoring peptide peak intensities 
 may also provide an indirect indication of impaired lc performance 
 and peak broadening that typically goes along with a lowering of peak 
 intensities .
alternatively , decreased peak intensities and peak areas 
 may suggest either an incorrect amount of injected sample or sample 
 loss on the part of the lc , or a problem on the part of the ms such 
 as less effective ionization or loss of ions before reaching the detector 
 or loss of detector sensitivity .
electrospray quality 
 and stability can be evaluated indirectly by its impact on ms and ms / ms ion injection times , number of acquired ms and ms / ms scans , and suppressed ( or absent ) peak intensities 
 and areas of peptides on simpatiqco  peptide lists  . 

all these metrics can be plotted over time to help provide information 
 including the spray quality and stability .
in addition , ms and ms / ms tic as well as the ratio  ( % ) of target value  
 can be plotted over the retention time for individual raw files .
high 
 ion injection times , low number of ms and ms / ms scans , 
 a low tic , and the presence of fluctuations of tic between ms scans can suggest problems with electrospray , and will often 
 prompt an exchange of the electrospray emitter .
high injection times , 
 low number of ms and ms / ms scans , and low tic can also 
 be a sign of electron multiplier gain calibration being out of calibration .
for the qc 1 sensitivity sample that includes cytochrome c , bsa , and 10 phosphopeptides , plotting the sequence coverage 
 of bsa is recommended in the first place , as this value reflects overall 
 lc  ms system sensitivity .
the status of a system may be considered 
 as appropriate for an analysis of , e.g. , routine samples ( such as 
 the identification of proteins from immunoprecipitation or tandem 
 affinity purification experiments ) when the bsa sequence coverage 
 is adequate .
qc 1 samples can be measured with disparate types of 
 fragmentation techniques , e.g. , collision - induced dissociation ( cid ) , 
 electron transfer dissociation ( etd ) , or higher energy collisional 
 dissociation ( hcd ) depending on the methods used in the ensuing regular 
 samples .
time course plots suggest that the average ms ion injection time ( calculated over the entire qc 1 run ) may also 
 constitute a sensitive indicator of ms system performance .
an increase 
 of the average ms ion injection time can be reverted by 
 appropriate actions such as instrument calibration , or cleaning of 
 the contaminated parts of the ion optics ( figure 2 ) . in a series of several
qc 1 runs the average ms ion injection time already increased while the bsa sequence coverage 
 still remained within the range of stochastic variation , suggesting 
 that in certain situations the average ms ion injection 
 time may constitute an even more sensitive indicator of subtle changes 
 before a drop in sensitivity becomes apparent ( figure s-3 in the supporting information ) .
we therefore monitor 
 both bsa sequence coverage and the ms ion injection time 
 closely to schedule the appropriate maintenance or ms system tests 
 for an evaluation of possible contamination of the ion path when the 
 average ms ion injection time starts to rise or when the 
 bsa sequence coverage drops below normal .
as qc 1 sensitivity 
 samples are analyzed once or even more than once per day , the metrics 
 derived from these samples reflecting lc performance , electrospray , 
 and ms sensitivity are monitored closely .
the qc 2 speed and 
 performance sample , a hela digest , is analyzed at least once per week 
 and in addition before an analysis of complex samples that require 
 optimum system speed .
similar to qc 1 samples , qc 2 runs can be performed 
 separately for each type of fragmentation method used for measuring 
 regular samples on the respective instrument ( e.g. , cid , etd , or hcd ) . 

the qc 2 sample is a complex protein digest , providing complementary 
 information with regard to system acquisition speed and performance 
 ( figure s-1 a in the supporting information ) .
the number of peptide - spectrum matches and identified proteins 
 indicate whether the lc 
hence these numbers constitute 
 a sensitive indicator of overall lc  ms system performance for 
 such samples .
ms and ms / ms ion injection times can 
 also be plotted over the retention time of a qc 2 raw file to determine 
 how frequently the ion injection time reached the maximum value , suggesting 
 that the agc target value might not have been reached for the respective 
 scan .
in addition simpatiqco calculates  ( % ) of target value  , 
 an estimator of the number of ion charges present in each scan divided 
 by the agc target value .
finally , 
 the average lock - mass deviation may serve as an indicator when the 
 next mass calibration should be scheduled .
qc runs are repeated 
 when outliers are observed so that impaired system performance should 
 become apparent as a series of outliers . whenever the pattern of the 
 time course of qc metrics suggests suboptimal performance ,
simpatiqco also assists with this 
 task . for instance , one may wish to plot the lock mass deviation , ms tic , the number of psms identified 
 per minute , and the m / z of triggered 
 precursor ions over the course of the qc run ( figure 3 ) .
lock mass deviation from a qc 1 sensitivity run ( cid ) : peculiar 
 pattern , plateaus indicate that lock mass was not detected ( a ) .
tic 
 from a qc 2 performance run ( etd ) : considerable fraction of tic between 
 180 and 200 min ( b ) .
different qc 2 performance run ( etd ) : psms per 
 minute ( c ) and m / z of triggered 
 precursors ( d ) plotted over the entire retention time range of the 
 raw file .
although simpatiqco can assist in finding the reason 
 of suboptimal system performance , in many situations lc - specific and 
 ms - specific system evaluation tests need to be performed to find out 
 the actual cause . in severe cases ,
prolonged ms ion injection 
 times are observed even when spraying calibration mixture solution 
 with the esi source mounted .
simpatiqco also offers the possibility 
 to upload such data , for instance 100 off - line acquired ms scans of a calibration mixture . a special type of qc - sample  run - type  

can be defined for such data and configured so that the data is not 
 searched with mascot .
when such data sets are uploaded on a regular 
 basis , simpatiqco allows convenient monitoring of the time course 
 of average ion injection times of ms scans of a calibration 
 mixture acquired off - line with the esi source , which should be below 
 0.1 ms on velos orbitrap instruments ( figure s-4 in the supporting information ) . among ms - specific 
 system evaluation procedures , the so - called  multipole flight 
 time evaluation  test , now called the  source optics 
 flight time evaluation  test , along with the
mp0 flight 
 time evaluation  test , can help provide an indication as to 
 whether there is a need to clean some element of the source ion optics 
 ( including the s - lens , exit lens , mp00 , lens 0 , or mp0 ) . in cases 
 where cleaning of the source optics does not lead to a sustained improvement 
 of the results of the qc metrics monitored by simpatiqco
, other parts 
 of the ion optics can be considered as a cause . in this case
another 
 system evaluation procedure called the  ion optics charging 
 evaluation  can help pinpoint a contaminated device along the 
 entire optics path including the exit lens , mp00 , lens 0 , mp0 , lens1 , 
 mp1 , gate lens , front lens , and center lens , by sequential exposure 
 to a negative ion beam .
the center lens between the high and low pressure 
 cells can also be evaluated via the  transfer efficiency evaluation  
 or  transfer lens calibration check  .
notably , appropriate 
 tuning of multipole offsets particularly with regard to mp0 can have 
 a strong effect on resisting contamination effects . with tune 2.7 
 sp1 , inadvertent setting of low voltages due to inappropriate
tuning 
 is prevented to ensure robust ion transmission even in case of moderate 
 contamination .
the first samples analyzed after starting the 
 lc  ms system again following troubleshooting and maintenance 
 procedures should always be further qc samples . at times , more than 

one cleaning approach may be required until the source of impaired 
 system performance can be identified and resolved ( figure 2 ) , which should then become apparent as long - term 
 sustained optimal performance , e.g. , with regard to bsa sequence coverage 
 and ms ion injection times and other performance metrics .
we developed simpatiqco ( simple automatic quality control ) , a software that aids 
 the successive and routine surveillance of sensitivity , speed , and 
 other qc performance metrics of lc  ms systems .
the software 
 is designed for use with ltq orbitrap , q - exactive , ltq ft , and ion 
 trap instruments manufactured by thermo scientific .
simpatiqco incorporates 
 data extraction , ms / ms spectra search , and storage of results within 
 a postgresql database coupled to an apache webserver , as well as calculation 
 of qc metrics that can be visualized remotely from a web browser client . 

taken together the available qc metrics allow monitoring of each step 
 of data acquisition .
the time course of qc metrics can be plotted 
 over a background colored in green , yellow , or red reflecting optimal 
 or impaired system performance as learned from the uploaded data by 
 calculation of robust statistics ( figure 2 and 
 figure s-1 in the supporting information ) .
simpatiqco also offers further 
 features such as plots of the number of psms per minute , lock mass 
 deviation , peptide elution times , apex intensities , peak widths , and 
 areas that aid operators in tracking possible causes ( figure 3 and figures s-1 and s-4 in the supporting information ) .
the benefit of monitoring the time 
 course of qc metrics currently becomes more widely recognized .
for 
 instance the association of biomolecular resource facilities ( abrf ) 
 prg 2012 study aims at evaluating within - laboratory instrument reproducibility 
 ( study announcement on the webpage http://www.abrf.org/ ) .
simpatiqco may be used by research laboratories and core facilities 
 alike to achieve this goal .
the software package is available free 
 of charge and can be downloaded from the webpage http://ms.imp.ac.at/ together with installation instructions and a manual .
we hope that 
 simpatiqco will help proteomics laboratories achieve and maintain 
 optimal quality of proteomics data acquisition .
 while the performance of liquid chromatography ( lc ) and 
 mass spectrometry ( ms ) instrumentation continues to increase , applications 
 such as analyses of complete or near - complete proteomes and quantitative 
 studies require constant and optimal system performance . 
 for this 
 reason , research laboratories and core facilities alike are recommended 
 to implement quality control ( qc ) measures as part of their routine 
 workflows . 
 many laboratories perform sporadic quality control checks . 
 
 however , successive and systematic longitudinal monitoring of system 
 performance would be facilitated by dedicated automatic or semiautomatic 
 software solutions that aid an effortless analysis and display of 
 qc metrics over time . 
 we present the software package simpatiqco ( simple automatic quality control ) designed 
 for evaluation of data from ltq orbitrap , q - exactive , ltq ft , and 
 ltq instruments . 
 a centralized simpatiqco server can process qc data 
 from multiple instruments . 
 the software calculates qc metrics supervising 
 every step of data acquisition from lc and electrospray to ms . for 
 each qc metric 
 the software learns the range indicating adequate system 
 performance from the uploaded data using robust statistics . 
 results 
 are stored in a database and can be displayed in a comfortable manner 
 from any computer in the laboratory via a web browser . 
 qc data can 
 be monitored for individual lc runs as well as plotted over time . 
 
 simpatiqco thus assists the longitudinal monitoring of important qc 
 metrics such as peptide elution times , peak widths , intensities , total 
 ion current ( tic ) as well as sensitivity , and overall lc  ms 
 system performance ; in this way the software also helps identify potential 
 problems . 
 the simpatiqco software package is available free of charge . 
Introduction
Methods
Results and Discussion
Conclusion