Space Charge Effects When Using Max AGC settings
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Gautam Saxena
Feb 27, 2017, 2:01:35 PM2/27/17
to ProteomicsQA
Hi, On a Fusion instrument, we've set the AGC setting to 5e6 (max value), which is about 20x higher than the default/recommended value of 1e5. Our questions are:
- Does space charge effect resolution in addition mass accuracy? If so, how would you characterize/describe this effect?
- What's best guess as to the impact on mass accuracy and what does it depend on? Specifically, is the mass accuracy something that is linearly dependent on total ions, or ion intensity of a 'nearby' ion, or the current ion's intensity, or some combination of all 3? (So far, the best article/paper I found on the correction needed is here, but it appears to imply that resolution is not affected (since they're silent on that topic) and that mass accuracy is affected as a function of AGC:
- To what extent can/should external calibration work to correct for mass accuracy (based on your hunches/experiences etc.)?
- Does this very high AGC also impact sensitivity in a negative way because ions with similar mz might collide?
On a completely different note, we're looking for part-time help with various bioinformatics programming needs -- is it acceptable to post for such a request (with relevant details) in this forum?
David Hollenstein
Feb 28, 2017, 7:56:53 AM2/28/17
to ProteomicsQA
Hi, this is actually a quite interesting question. I do have some minor
knowledge about the effects, but I don't want to speculate too much about it.
I will try to do some small experiments to show the effects when I have time,
maybe tomorrow. I will then post the results here.
What I know so far is that if two ions are close together in the m/z space,
the signals actually merge when you collect too many of the ions. So you
definitely have a loss in resolution, at least under certain conditions.
About your AGC target values, is this for MS1 or MSn? I think it depends on
the complexity of your sample whether an MS1 AGC setting of 5e6 is
appropriate. If you have a complex and evenly distributed mixture of abundant
ions, I would guess that none reach the numbers that cause space charge
effects. On the other hand if you measure the peptides of one single protein
or collect MS2 spectra, I would guess that single ions can actually reach a
critical number where these effects start to appear.
If you are talking about MS1 AGC targets, an increase of signal abundance by a
factor of 50 (1e5 to 5e6) will quite noticeable increase your sensitivity.
However, I don't think that under normal circumstances you can even reach
these numbers in an MSn scan. And I doubt that it will substantially increase
the quality of the fragment spectra, since spectra with 1e5 already tend to be
quite good, but I might be wrong here.
About your job offer, I think this would be the right place to post it.
http://wiki.proteomics-academy.org/Jobs
Best regards
David
knowledge about the effects, but I don't want to speculate too much about it.
I will try to do some small experiments to show the effects when I have time,
maybe tomorrow. I will then post the results here.
What I know so far is that if two ions are close together in the m/z space,
the signals actually merge when you collect too many of the ions. So you
definitely have a loss in resolution, at least under certain conditions.
About your AGC target values, is this for MS1 or MSn? I think it depends on
the complexity of your sample whether an MS1 AGC setting of 5e6 is
appropriate. If you have a complex and evenly distributed mixture of abundant
ions, I would guess that none reach the numbers that cause space charge
effects. On the other hand if you measure the peptides of one single protein
or collect MS2 spectra, I would guess that single ions can actually reach a
critical number where these effects start to appear.
If you are talking about MS1 AGC targets, an increase of signal abundance by a
factor of 50 (1e5 to 5e6) will quite noticeable increase your sensitivity.
However, I don't think that under normal circumstances you can even reach
these numbers in an MSn scan. And I doubt that it will substantially increase
the quality of the fragment spectra, since spectra with 1e5 already tend to be
quite good, but I might be wrong here.
About your job offer, I think this would be the right place to post it.
http://wiki.proteomics-academy.org/Jobs
Best regards
David
Gautam Saxena
Feb 28, 2017, 7:52:18 PM2/28/17
to ProteomicsQA
Hi David,
I'm glad you like the question! Actually, I think you might find it even more interesting if you knew some background info. Specifically, we are exploring this question not for DDA runs (where arguably it would not be as valuable, as you pointed out in your post below), but we are exploring this in DIA. In DIA, unlike DDA, the more sensitive we can make the MS2 DIA windows (plus even the MS1 window, though that's less important for us), the better. Specifically, if we can increase the AGC by 50x, then in theory, our sensitivity is 50x more as well, which is a non-trivial benefit. In fact, one could argue that unless the mass accuracy loss is ridiculously high at the point that the mz values are effectively unusable, I could see someone arguing that it is (far?) better to have 50x sensitivity and still at least see the signals (even if the mass accuracy isn't the super awesome under 5 PPM) rather than having the super awesome accuracy (such as under 1 or 5 PPM) but never seeing those low abundance signals to begin with....
What I don't know, though, is what level of mass accuracy do we lose when we go from the recommended AGC values of 1e5 to the maximum possible value of 5e6. Moreover, it's unclear to me under what circumstances we lose this mass accuracy? That is, is this loss in mass accuracy across the board? Or, it more pronounced for large signals/small signals or only when a large signal is next to a small signal? And, can external calibration correct for this issue in DIA MS2 signals on the Fusion?
Finally, at least on the latest Thermo Tune 2.1 software, it would appear that Thermo does some sort of calibration to account for the AGC level. As I recall, if you look in the detailed section for a given spectra, at the very bottom it'll list some sort of correction that it applies due to the AGC level. So, my other question are: is that calibration sufficient or would external calibration have benefits (as implied by the paper)? And, does Thermo Tune 2.1 calibration due to AGC work even if we've turned off the IC (internal calibrant)? (Thermo now recommends disabling IC for the DIA runs, since it adds ~20ms/DIA window, so if you have say 40 windows, it significantly increases cycle time if you include the IC.) Sorry for all my questions, but once I started going, I realized I had a bunch. Feel free to respond to only those that you're comfortable responding to.
Thanks in advance, David.
I'm glad you like the question! Actually, I think you might find it even more interesting if you knew some background info. Specifically, we are exploring this question not for DDA runs (where arguably it would not be as valuable, as you pointed out in your post below), but we are exploring this in DIA. In DIA, unlike DDA, the more sensitive we can make the MS2 DIA windows (plus even the MS1 window, though that's less important for us), the better. Specifically, if we can increase the AGC by 50x, then in theory, our sensitivity is 50x more as well, which is a non-trivial benefit. In fact, one could argue that unless the mass accuracy loss is ridiculously high at the point that the mz values are effectively unusable, I could see someone arguing that it is (far?) better to have 50x sensitivity and still at least see the signals (even if the mass accuracy isn't the super awesome under 5 PPM) rather than having the super awesome accuracy (such as under 1 or 5 PPM) but never seeing those low abundance signals to begin with....
What I don't know, though, is what level of mass accuracy do we lose when we go from the recommended AGC values of 1e5 to the maximum possible value of 5e6. Moreover, it's unclear to me under what circumstances we lose this mass accuracy? That is, is this loss in mass accuracy across the board? Or, it more pronounced for large signals/small signals or only when a large signal is next to a small signal? And, can external calibration correct for this issue in DIA MS2 signals on the Fusion?
Finally, at least on the latest Thermo Tune 2.1 software, it would appear that Thermo does some sort of calibration to account for the AGC level. As I recall, if you look in the detailed section for a given spectra, at the very bottom it'll list some sort of correction that it applies due to the AGC level. So, my other question are: is that calibration sufficient or would external calibration have benefits (as implied by the paper)? And, does Thermo Tune 2.1 calibration due to AGC work even if we've turned off the IC (internal calibrant)? (Thermo now recommends disabling IC for the DIA runs, since it adds ~20ms/DIA window, so if you have say 40 windows, it significantly increases cycle time if you include the IC.) Sorry for all my questions, but once I started going, I realized I had a bunch. Feel free to respond to only those that you're comfortable responding to.
Thanks in advance, David.
-Regards,
Gautam
Gautam
David Hollenstein
Mar 1, 2017, 2:38:59 AM3/1/17
to ProteomicsQA
Hi Gautam,
I think that you have to do some experiments to explore the effects of
increasing your AGC target, in order to be able to decide what is most
beneficial. Besides the observed effect size, and the question whether you
think it is tolerable, one important point to consider is if your software can
handle data with these effects, or if it starts producing wrong results.
Regarding the sensitivity increase. It is an interesting idea, but I'm still
not sure if you are actually able to collect such a large amount of ions
during the small time frame of one swath window. Since I never did DIA myself
I'm not sure how the method looks, especially on a fusion. I guess you still
analyze the MSn scans with the Orbitrap for higher resolution? What are your
resolution settings and what ion injection times (IIT) do you allow?
You can estimate to maximum gain in signal intensity that you can achieve by
looking at the actual IITs, and dividing the maximum IIT by the observed ones.
If you already reach the maximum, increasing the AGC will make no difference.
Then you could increase the max IIT, which could increase your cycle time
depending on the other settings, or inject more sample, which will result in
overloading your LC at one point.
Best regards
David
I think that you have to do some experiments to explore the effects of
increasing your AGC target, in order to be able to decide what is most
beneficial. Besides the observed effect size, and the question whether you
think it is tolerable, one important point to consider is if your software can
handle data with these effects, or if it starts producing wrong results.
Regarding the sensitivity increase. It is an interesting idea, but I'm still
not sure if you are actually able to collect such a large amount of ions
during the small time frame of one swath window. Since I never did DIA myself
I'm not sure how the method looks, especially on a fusion. I guess you still
analyze the MSn scans with the Orbitrap for higher resolution? What are your
resolution settings and what ion injection times (IIT) do you allow?
You can estimate to maximum gain in signal intensity that you can achieve by
looking at the actual IITs, and dividing the maximum IIT by the observed ones.
If you already reach the maximum, increasing the AGC will make no difference.
Then you could increase the max IIT, which could increase your cycle time
depending on the other settings, or inject more sample, which will result in
overloading your LC at one point.
Best regards
David
Vladimir Gorshkov
Mar 1, 2017, 5:46:06 AM3/1/17
to ProteomicsQA
Hi Gautam,
There is an AGC value correction that is performed routinely every time one perform Orbitrap mass calibration (it spans up to 1e6) and the software fits a linear calibration into actual data. It indeed looks quite linear. This data is used to calculate the factors you see in the scan header. Thus, I guess you won't sacrifice the mass accuracy so much if you use 5e6, however, the easiest way to check is to inject calmix at 5e6 and acquire some scans. I guess you can get < 10 ppm.
A colleague of mine is working with histone proteins and he observes the degradation in resolution starting with 2e5. It is, of course, somewhat extreme case, since ions there are quite large (in size and charge) with small m/z differences. Smaller and less charged ions should be more tolerant to coalescence.
We did some tests and observed that independently of AGC target (we tested more common values 1e4 - 1e6) and intensity there is# about 4 orders of magnitude dynamic range in individual Orbitrap scans. Thus, there is a chance that the increase in low abundant ions won't be so great even with the highest AGC setting.
Best regards,
Vladimir
There is an AGC value correction that is performed routinely every time one perform Orbitrap mass calibration (it spans up to 1e6) and the software fits a linear calibration into actual data. It indeed looks quite linear. This data is used to calculate the factors you see in the scan header. Thus, I guess you won't sacrifice the mass accuracy so much if you use 5e6, however, the easiest way to check is to inject calmix at 5e6 and acquire some scans. I guess you can get < 10 ppm.
A colleague of mine is working with histone proteins and he observes the degradation in resolution starting with 2e5. It is, of course, somewhat extreme case, since ions there are quite large (in size and charge) with small m/z differences. Smaller and less charged ions should be more tolerant to coalescence.
We did some tests and observed that independently of AGC target (we tested more common values 1e4 - 1e6) and intensity there is# about 4 orders of magnitude dynamic range in individual Orbitrap scans. Thus, there is a chance that the increase in low abundant ions won't be so great even with the highest AGC setting.
Best regards,
Vladimir
Gautam Saxena
Mar 1, 2017, 8:37:31 AM3/1/17
to David Hollenstein, ProteomicsQA
Hi David,
Regards,
To answer your question: we do not increase cycle time, since we keep the IIT fixed to be 54 ms, which is under the time time it takes for the orbi to perform a scan at resolution 30k. However, we observed that both for MS1 and MS2, the fill times were far less than 54 ms when the AGC settings was set to the normal 1e4; even at 5e6, we often didn't hit the 54 ms IIT limit. So, in "theory", increasing AGC from 1e4 to 5e6 provides more signal (and certainly the MS spectra looks far more busy/crowded)....
Thanks in advance, David.
Regards,
Gautam
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Gautam Saxena
President & CEO
Integrated Analysis Inc.
Making Sense of Data.™
Biomarker Discovery Software | Bioinformatics Services | Data Warehouse Consulting | Data Migration Consulting
(301) 760-3077 office
(240) 479-4272 direct
(301) 485-7364 fax
(301) 485-7364 fax
Gautam Saxena
Mar 1, 2017, 8:37:51 AM3/1/17
to Vladimir Gorshkov, ProteomicsQA
Hi Vladmir,
Ok, your point regarding dynamic range is something that I hadn't considered. So if I understand you correctly, what you are suggesting is that the orbitrap's internal dynamic range ends up being the limiting factor. Specifically, you're suggesting that the orbitrap in a Fusion (not Lumos in our case) would likely have a dynamic range of ~4 order of magnitude, so if the scenario is that we have one "really big peak, eg intensity of say 2e6" and say lots (eg dozens) of little peaks (intensity of 5e1), we won't see those little peaks even with an AGC of 5e6 due the orbitrap's dynamic range limit; however, if the scenario were that we have lots of "medium sized peaks", eg, ~10 peaks of intensity 2e5 (so the sum is roughly equivalent of one big peak of 2e6), then having the max AGC setting of 5e6 would be beneficial for those low abundant peaks, as we would now be able to see the dozens of little peaks (since in this scenario we are *both* within the orbitrap's dynamic range and also within the AGC limit.) Is that right?
Also, just to clarify, when you referred to observed resolution issues for the histone study, you were referring to using histone proteins in the MS (top down) and not peptides from histone (bottom-up), right? That is, the MS1 was seeing ions of ~10,000 Da (very large ions) with charges much higher than the standard charge +2 to +5 we see on peptides, right? (I've never analyzed data from top-down ms.) If that is the case, would it be too far a stretch to say that even at AGC of 5e6, for the 'normal' small ions we see with bottom-up MS proteomics, the resolution will NOT be affected, even if the mass accuracy is shifted? (But, presumably, the shift is linear or even non-linear, but at least it can be expressed as a function of some sort that could, in theory, be determined from observed data using an external calibration fitting function of some sort....)
Thanks in advance, Vladmir.
Regards,
Gautam
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Vladimir Gorshkov
Mar 2, 2017, 5:35:55 AM3/2/17
to ProteomicsQA, hom...@gmail.com, gsa...@i-a-inc.com
Hi Gautam,
Yes, that is what we observe. Increasing the AGC rather scales up all intensities in the spectrum proportionally. If the highest peak in the scan has intensity, say, 1e6 the lowest observable will be about 1e2. With the twice bigger number of ions the major one will become 2e6, but the smallest observable will be 2e2 instead. We have this observation both on QE-HF and Fusion(Lumos), both using high-field D20 orbitrap. It looks like the design limitation of the Orbitrap itself, or the C-trap, probably.
Please, see the figures attached, the plots show mean intensity (log10 transformed) and the ratio of log10 transformed maximal and minimal intensities in the spectrum, i.e. the dynamic range in orders.
Yes, I was talking about top-down applications (i.e. ~10 - 15 kDa molecules with charges of 15 and higher). Well, honestly, I can not be absolutely sure if the effect is so much severe in the case of usual peptide ions, since 1e6 is quite regular I guess the effect should not be severe (if any). However, I think it is definitely something to keep an eye on.
I think you should definitely use additional mass calibration, especially if you aim at DIA application, that might aid in deciphering the spectrum. And it dosen't "cost" much after all.
Best regards,
Vladimir
David Hollenstein
Mar 2, 2017, 1:51:06 PM3/2/17
to ProteomicsQA, hollenst...@gmail.com, gsa...@i-a-inc.com
Hi Gautam!
I managed to do some small experiments yesterday. I focused the MS1 isolation
window around prominent ions from the ambient air +/- approximately 5 m/z.
Then recorded 20 scans for AGC target values of 1e2, 1e3, 1e4, 1e5 and 1e6. I
also allowed a pretty long IIT (up to 2 seconds) in order to be able to
collect enough ions of the higher mass ions, which do have a lower intensity.
I only analyzed the mono isotopic peak of each isotope cluster.
What I call the "recorded intensity" is "intensity value / iit (in seconds)"
and corresponds to the amount of collected ions. As you can see in the
attached graph, it looks like that not only the m/z accuracy but also the
accuracy of the measured intensity is negatively influenced by space-charge
effects. At least in this example the effects start to become apparent around
a recorded intensity of 1e5.
Taken together with the information from Vladimir I would suggest that you
simply test it with a reasonably realistic setup. You could for example
measure a sample that corresponds to the complexity and intensity of your
normal samples. Then you make a method where you record each swath window
twice in a row, the first one is recorded with your normal AGC target value
and the second with the increased one. Then you can compare all identical ions
between the adjacent scan pairs, and check whether their intensity and m/z
values change in an intensity dependent manner. In addition you can analyze
whether there are more ions in the higher AGC scans. If the software that you
are using for analysis accepts mzML files, you can also generate two files,
one containing only the low AGC scans and one containing the high AGC scans.
Then you compare the results of your analysis pipeline for both mzML files.
Best regards,
David
I managed to do some small experiments yesterday. I focused the MS1 isolation
window around prominent ions from the ambient air +/- approximately 5 m/z.
Then recorded 20 scans for AGC target values of 1e2, 1e3, 1e4, 1e5 and 1e6. I
also allowed a pretty long IIT (up to 2 seconds) in order to be able to
collect enough ions of the higher mass ions, which do have a lower intensity.
I only analyzed the mono isotopic peak of each isotope cluster.
What I call the "recorded intensity" is "intensity value / iit (in seconds)"
and corresponds to the amount of collected ions. As you can see in the
attached graph, it looks like that not only the m/z accuracy but also the
accuracy of the measured intensity is negatively influenced by space-charge
effects. At least in this example the effects start to become apparent around
a recorded intensity of 1e5.
Taken together with the information from Vladimir I would suggest that you
simply test it with a reasonably realistic setup. You could for example
measure a sample that corresponds to the complexity and intensity of your
normal samples. Then you make a method where you record each swath window
twice in a row, the first one is recorded with your normal AGC target value
and the second with the increased one. Then you can compare all identical ions
between the adjacent scan pairs, and check whether their intensity and m/z
values change in an intensity dependent manner. In addition you can analyze
whether there are more ions in the higher AGC scans. If the software that you
are using for analysis accepts mzML files, you can also generate two files,
one containing only the low AGC scans and one containing the high AGC scans.
Then you compare the results of your analysis pipeline for both mzML files.
Best regards,
David
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--Gautam SaxenaPresident & CEOIntegrated Analysis Inc.Making Sense of Data.™Biomarker Discovery Software | Bioinformatics Services | Data Warehouse Consulting | Data Migration Consulting(301) 760-3077 office(240) 479-4272 direct
(301) 485-7364 fax
David Hollenstein
Mar 2, 2017, 3:44:39 PM3/2/17
to ProteomicsQA, hollenst...@gmail.com, gsa...@i-a-inc.com
Hi,
I forgot to mention that the measurements were performed on
a Thermo Fusion instrument.
Gautam Saxena
Mar 2, 2017, 4:33:00 PM3/2/17
to ProteomicsQA, hollenst...@gmail.com, gsa...@i-a-inc.com
Cool, we're digesting your results right now...that said, what resolution setting did you use?
David Hollenstein
Mar 3, 2017, 5:39:52 AM3/3/17
to ProteomicsQA, hollenst...@gmail.com, gsa...@i-a-inc.com
Hi, the resolution was 120k.
Gautam Saxena
Mar 3, 2017, 3:13:35 PM3/3/17
to ProteomicsQA, hollenst...@gmail.com, gsa...@i-a-inc.com
Hi David & Vladimir: Your responses were excellent and caused some good discussion in our team. We're now running a hela sample 3 times (DDA mode only) on a Fusion with settings of 1e5, 1e6, and 5e6 for AGC, and then running it through PD, and then attempting to understand the MS1 ppm error. We're expecting the MS person to do so by Monday, so hopefully will have some results to share shortly thereafter.
Gautam Saxena
Mar 14, 2017, 2:04:51 PM3/14/17
to ProteomicsQA, hollenst...@gmail.com, gsa...@i-a-inc.com
We ran 3 DDA runs (hela digest) at 3 different AGC levels (1e5, 1e6, 5e6) and the raw results can be found here: https://az.box.com/s/j72qyluzuxl1v3degxqwx0mwzzuyh260 (I'm sharing before we've completed our analysis and I prefer to NOT lead everybody, but it would appear, at least at first, that 1e6 does well and 5e6 does well from a # of PSM perspective (see MassAccuracy_MS1_level_2_datasets.pptx for quick-and-dirty graphical summary; also, ignore trial 1 results and focus on trial 2 results). I think that with DIA data -- unlike DDA data -- 5e6 data might do quite well.....) If anyone wants to review these results and share their findings too, that would be appreciated. I have not yet parsed and re-analyzed the PSM txt files. (I just reviewed the ppt.)
David Hollenstein
Mar 14, 2017, 3:49:48 PM3/14/17
to ProteomicsQA, hollenst...@gmail.com, gsa...@i-a-inc.com
Hi,
I just had a quick look at the PSM files, I found that there is an IIT columen included.
However, since this is DIA I don't know how exactly these "PSMs" are generated.
But when looking at the IIT values, and at the scans they represent, it looks to me
that increasing the AGC only had a minor effect. This is because in the 1e5
experiments around 95% of all "PSMs" already reached the max IIT, i.e. in the
experiments 1e6 and 5e6 only 5% of the PSMs could have had an increase in the
collected ions. Altough the system would be allowed to collect higher amounts of
ions, it does not have enough time to do so. Thus you would also expect to only
see a minor increase in the number of identified "PSMs". The experiments 1e6
and 5e6 should look identical on the PSM level, both have 100% max IIT reached.
However, since the entries in the PSM files are not really scans, I guess it would
make more sense to do this analysis at the spectrum level.
Greets
David
I just had a quick look at the PSM files, I found that there is an IIT columen included.
However, since this is DIA I don't know how exactly these "PSMs" are generated.
But when looking at the IIT values, and at the scans they represent, it looks to me
that increasing the AGC only had a minor effect. This is because in the 1e5
experiments around 95% of all "PSMs" already reached the max IIT, i.e. in the
experiments 1e6 and 5e6 only 5% of the PSMs could have had an increase in the
collected ions. Altough the system would be allowed to collect higher amounts of
ions, it does not have enough time to do so. Thus you would also expect to only
see a minor increase in the number of identified "PSMs". The experiments 1e6
and 5e6 should look identical on the PSM level, both have 100% max IIT reached.
However, since the entries in the PSM files are not really scans, I guess it would
make more sense to do this analysis at the spectrum level.
Greets
David
Gautam Saxena
Mar 14, 2017, 4:11:29 PM3/14/17
to David Hollenstein, ProteomicsQA
Cool David. Just a minor point (which may or may not change your final analysis -- I'm not sure). These results were for DDA, not DIA, since the MS person could easily run DDA. We are using these 'routine' DDA runs to "infer" what would happen in DIA experiments.
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David Hollenstein
Mar 14, 2017, 4:27:40 PM3/14/17
to ProteomicsQA, hollenst...@gmail.com, gsa...@i-a-inc.com
Hi,
sorry. Somehow I missed that, probably because I was expecting that you did DIA =)
I think this shows you that the amount of sample you inject, in combination with your
max fill time, does not allow you to strongly increase the number of collected ions just
by changing the AGC target value.
Probably you either have to load more sample, or increase the fill times to see a
bigger effect, but with increased fill times you record less MS2 scans in a DDA
setting...
Greets
sorry. Somehow I missed that, probably because I was expecting that you did DIA =)
I think this shows you that the amount of sample you inject, in combination with your
max fill time, does not allow you to strongly increase the number of collected ions just
by changing the AGC target value.
Probably you either have to load more sample, or increase the fill times to see a
bigger effect, but with increased fill times you record less MS2 scans in a DDA
setting...
Greets
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