We have developed the visualization tool LogViewer to quickly and easily assess and evaluate the overall instrument's performance, as well as individual sample quality. LogViewer allows for visualization of diagnostic output data from log files generated by RawXtract4
and routine monitoring of important QC metrics. It displays those metrics that are important for a successful LC-MS/MS experiment: metrics that change during a routine analysis and are affected by user-specified parameters (e.g., ion-injection time, charge-state selection). The use of LogViewer to optimize instrument parameters using yeast lysate is illustrated and discussed in the following subsections.
MS- and MS2-Injection Times
The rate-determining step of an ion trap analytical cycle is the MS- and MS2-injection time, the time it takes for precursor (MS) or fragment ions (MS2) to fill the trap. In a given setup, short MS (and MS2) ion-injection times are thus indicative of an optimized spray. MS (and MS2) ion-injection times are displayed as histograms to monitor overall performance or versus retention time, to display variations during an analysis. Although ionization efficiency has an effect on MS and MS2, we found short MS ionization times to indicate mostly optimized spray conditions. On an Orbitrap Classic or LTQ-FT collision-induced dissociation (CID) experiment, we generally see for automatic gain control target values of 1 × 106 (MS) and 5 × 103(MS/MS) ion-injection times of <50 ms in an optimized spray throughout the analysis. shows a screenshot of the MS histogram showing a median ion-injection time of 5 ms. It should be noted that analogous values for an Orbitrap Velos should be considerably lower, as the Orbitrap Velos has a significantly improved ion transmission. During a spray-optimized analysis, larger variations may be observed before the sample is actually loaded on the column, indicating a lack of ionizable peptides at this time. Spray instabilities are detected with increased mean-injection times in the histograms and in the ion-injection time versus retention time display. This feature can also be helpful when samples are analyzed unsupervised (overnight and over weekends), and performance has decreased over this time period. If the spray has become unstable in the middle of a sequence, it can detect exactly when this happened and which samples need to be reanalyzed, as the occurrence of spray instability during a LC run can be easily determined using LogViewer.
FIGURE 2 MS ion histogram. An optimized sample showed a median-injection time of 5 ms. Additional screenshots of all parameters of an optimized sample are available in the Supplemental information.
This metric was also useful to identify valve-closing irregularities on a previously used UltraPerformance LC (UPLC) pump. These irregularities presented themselves as spikes ca. every 25 min () with the median-injection time still on target (below 20 ms). Here, the benefit of visualization tools, such as LogViewer, becomes apparent immediately. Even an untrained eye recognizes the spiked pattern and can start to investigate the underlying reason. In addition to optimized spray conditions, short MS2-injection times indicate optimization of sample load to the chosen instrument parameters.
Injection time versus retention time shows spikes ca. every 25 min, revealing valve-closing irregularities of the UPLC pump.
Dependent Scan Histograms
When surveying the literature, it is often difficult to judge how many MS/MS data-dependent scans should be chosen for every precursor scan. For CID experiments, recommendations vary mostly between three and 10 data-dependent scans/precursor scan. When displaying the dependent scan number, we were able to identify performance problems when the frequency of dependent scans dropped as a result of inappropriate instrument settings (e.g., when the ACG targets for MS and MS2 were not optimized).
When peptides are ionized, their protonation status and thus, their charge states vary. It has been described previously5,6
that doubly charged ions fragment better in CID experiments than triply charged ions, wheras triply charged ions fragment better in electron transfer dissociation (ETD) experiments. In an effort to optimize for the preferential formation of doubly charged ions in CID, we experimented with different needle-tip materials (silica, coated and uncoated, metal). With the use of LogViewer, we found that the tip material influenced the generation of different charge states. A needle generating preferentially doubly charged ions was the New Objective PicoTip emitter with a P200P coating creating >70% doubly charged ions, followed by ca. 25% triply charged ions and few quadruply or higher charged ions. In contrast, uncoated emitters regularly produce less doubly charged ions (data not shown). In our optimized settings, we exclude singly charged ions, as many background ions are singly charged. Once optimized for the preferred charge state, unexpected run-to-run changes in charge-state distributions can be indicative of changes in sample composition (e.g., as a result of contamination, insufficient digestion).
M/Z Distribution and Histograms
Literature reports also often vary widely in the use of the M/Z range for the precursor scan (scan event 1). For instance, the CPTAC study recommended the use of 300–2000 M/Z. When monitoring the M/Z distribution, we noticed that for instance, yeast lysates rarely display M/Z above 1600. Thus, we limited the M/Z range to 300–1600 for yeast lysates. The narrower the M/Z window, the faster the scans can be performed, increasing the duty cycle and thus, the number of scans that can be analyzed in a given timeframe.
Mass Distribution and Histograms
The mass of a protonated peptide is calculated by multiplying its M/Z value with its charge state and subtracting its charge state times the proton mass. This additional information can be useful to identify incomplete digestion when an unexpected large number of polypeptides larger than 3500 Da are detected. Like most of the other metrics, this observation is dependent on the chosen LC and MS sample conditions. If, for instance, charge states are accepted or rejected, it will influence the observed mass distribution. A useful feature is the color coding in the mass distribution tab. Charge state 1 is displayed in red (not shown), charge state 2 is displayed in green, charge state 3 in blue, and charge state 4 and higher in cyan. Thus, an increase of singly or multiply charged ions can be visualized easily.
In the Xcalibur software, LC and MS settings can be theoretically matched so that every ion is supposedly analyzed only once. In practice, this is not the case. Even in our currently best optimizations, we observe an average of ca. four repeat analyses, as the observable peak width varies for different peptides. In addition, high abundant peptides may leach out over longer periods of time. Ideally, each peptide would be analyzed only once (at its most intense point in the chromatogram). If every peptide is analyzed more than once, the number of identified peptides could be increased significantly by just allowing the dynamic exclusion list to be long enough to accommodate every observable ion into this list. Changes can be monitored using LogViewer.
In addition to the individual plots, a summary table shows the number of MS events, number of MS2 events, number of MS ion-injection times, number of MS2 ion-injection times at the chosen maximum-injection times, and number of MS2 scans with charge state 1+, 2+, 3+, 4+, 5+, 6+, 7+.
Raw MS and Raw MS2
Finally, all scan numbers and their associated retention times, injection times, M/Z, and charge states (in case of MS2) are listed in a table to allow the specific interrogation of a certain scan.