This year we have the opportunity to purchase an HRMSD. We have an Agilent Infinity II 1260 HPLC in our lab and are planning to purchase a Q-TOF. I have experience with Agilent equipment (GC/LC/GC-MS SQ/LC-MS SQ), but not with HRMS.
We are planning to use this instrument for untargeted metabolomics. We are interested in searching for producers of new antimicrobial compounds, performing screening and identification of new antimicrobials of microbiological origin, and studying the biosynthesis of microorganisms.
So far, we have been offered two Agilent detectors:
1. 6546
2. Revident.
As I know HRMS produces huge amounts of data, and performing untargeted metabolomics workflow requires additional software to work with the data in case of untargeted analysis.
Now Agilent offers two sets of software, which is a bit confusing for me.
1. Let's call the first set "classic". It includes:
a. MassHunter Profinder (for Feature finding stage)
b. Mass Profiler Professional (MPP) (for Alignment and statistics)
i. ID Browser (module of MPP for Identification)
ii. PathWay Architect (optional MPP module for metabolite pathways buildings)
c. METLIN PCDL for LC/Q-TOF (database for metabolomics)
2. Let's call the second set "new" It includes:
a. software product - MassHunter Explorer, which, according to the manufacturer, combines all of the above software products into one.
b. ChemVista library manager with METLIN PCDL library
Questions:
1. Is the MassHunter Profinder standalone SW or is it part of MassHunter Qual or Mass Profiler Professional
2. Which one of the SW sets should be chosen? They do the same. But do they really do the same and have the same capability? Marketing? From my experience the early version of SW is quite restricted. For 6546 and the newest Revident Aglent recommends MassHunter Explorer.
3. To buy or not to buy:
a. I read that untargeted analysis has a huge community and freeware databases and SW for metabolite identification. Is METLIN PCDL library a MUST part of SW from the Vendor? Can I consider it as optional and use freeware DB?
b. The same question about ChemVista library manager?
4. By which SW/Databases do you realize your untargeted metabolomics workflow?
5. Any experience with the Revident model of Q-TOF. How far is it better/worth in metabolomics compared to 6546? Marketing?
a. Intelligent functions
b. Solutions for Tuning
6. Is the APCI source essential for untargeted metabolomics?
Thank you in advance.
Dear Andriy Ostapchuk,
Unfortunately, I can't tell you which device or software is the better choice for you. Basically, however, I would opt for the newer version, as most of them will be continued for a longer period of time. The Masshunter software offered includes many tools that can be used to quickly and unambiguously identify metabolomics. The add ons listed are additional tools to make the user's work easier. the use of the ads is a decision of the user and not a necessity. The Masshunter also offers the possibility to create and use your own databases.
We are currently using two Agilent 6530 Q-Tof in the field of degradation products of active pharmaceutical ingredients in aqueous media. The Masshunter version for our devices does not have any special ad ons and can easily detect unknown components in an analysis run and identify them as unknown connections.
The software can be trained to narrow down to new unknows quickly and securely. Whatever you choose, you will get good results with HRMS.
I wish you every success in your work.
Best Regards
Joachim Horst
Dear Andriy,
First, congratulation with your future instrument. I personally love Agilent 6546, I did not work with Revident though.
Second, in my opinion, the most important step in untargeted metabolomics by HRMS is to fully understand what it can and can't do. Basically, I think a researcher has to accept that untargeted metabolomcis will not provide meaningful results immediately. Also, one has to accept that a whole process is so complex (not difficult, but complex), that it will be sub-optimal for most of the chemicals of interest. Therefore, for example, I would not consider APCI as a must have source. In contrast, for the targeted analysis, APCI can be essential in some cases.
The software provided are kind of repeat each other (in my impression), so I ended up using mostly MPP for all tasks. I think it is quite nice, intuitive and powerful. I do prefer to perform post-processing data analysis using external soft, but build-in functions work well for the initial results.
I do recommend to have PCDL, it can save you a lot of time for the primary metabolites annotation on the fly. But you can use external soft and databases too, if PCDL is beyond your budget or so.
Maintenance of 6546 is acceptable. in my case I have to prepare tuning mix by combining purchased components, but I heard Agilent is going to (already did) provide prepared mixes. Cone cleaning is easy, the needle adjustment can be tricky, but hopefully you will not need it often. I also like Agilent's video instructions., but I strongly recommend you to make sure how good is Agilent support in your location. Problems are unavoidable, I had a great support in this cases, but it is in the US. I know that other locations can be different.
Good luck.
I agree with Albert Batushansky that PCDL is very helpful to recognize unknown components.
I also often work with File Open Actions to perform recurring data preparation while loading the dataset. I think in the course of time you will realize what can and cannot be done with the Mass Hunter. I also think it is helpful to have your own databases, where you can store spectra that can then be used for further samples. Even if the identity of the substance has not yet been clarified, it can be very helpful to store an unknown spectrum with an index of some kind so that it can be investigated whether it also appears in other samples under the same conditions or only sporadically.
Targeting the antimicrobial compound research, mainly low MWs indicates you will have a huge molecule pool as you indicated. Basic HR-MS systems (other than Orbitrap above 120.000 FWHM@ 200 Da) nowadays are not very effective in annotating low MW compounds due to the low FWHM of the TOF (res power; e.g. 40.000). I suggest you adding either drift tube IMS, SLIM (Mobilion) properties to your instrument to differentiate isomeric species before collision cell and observe 4D spectra. (I indicated the only configurations that can equipped with Agilent systems). There are many other options and vendors indeed.
You may also consider about the fragmentation hardware such as ETD/ECD beside the CID. This enables producing the alternative fragments and simplifies the molecule annotation. MS1 level database search reports many candidates and this needs either alternative MS/MS m/z values or MS1 (precursor) mobilogram.
Dear Joachim, Albert and Ismail?
I am very grateful to all of you for the answers! Honestly, I had only the slightest expectation that my question would be answered. But lucky me! Thanks for your time!
Dear Albert, could you please share which external software you use for the post-processing data stage?
I am not a big expert in HRMS, but as I know there is an open-source DB available in the web. Is there any freeware DB manager? Just interested in your opinion about the possible usefulness of Agilent ChemVista Data Base Manager.
Thanks again to everyone!
Andriy
Dear Andriy,
After peak-picking I usually export the numerical data to a spread-sheet software, and then use R.
There are many public databases available: KEGG, HMDB, ChemSpider. I did not work with Agilent ChemVista, so probably it has some important features. For example, it could provide straight-forward options for in-house library creation, but it would be better to ask Agilent directly. Agilent has very nice video instructions for many things, so maybe they can share with you the one about Vista. I am sure the will be benefits to have this software, but I don't think it will be anyhow critical.
The main benefit to have Agilent library (not necessarily integrated via Vista) is to have an ability to get a higher probability of metabolite annotation in kind of "live mode" just following they per-tested conditions. But you also can get MS/MS data from public sources like HMDB.
In my opinion, the most important part of metabolomics is to clearly understand what one wants to do. Because different "want" will require different approaches.