Hi Mohammed,

LC-MS and LC-MS/MS are the combination of liquid chromatography (LC) with mass spectrometry (MS). MS means you only analyze your precursor ion (as generated in the source) for example in an iontrap, a quadrupole or time-of-flight Massspec. MS/MS is the combination of two mass analyzers in one mass spec instrument. The first MS filters for the precursor ion followed by a fragmentation of the precursor ion with high energy and e.g. nitrogen gas. A second mass analyzer is then filtering for the product ions, generated by the fragmentation. This is usually done in a triple quadrupole MS (QQQ) or a QTOF. The advantages of MS/MS is the increased sensitivity (in the QQQ, due to reduction of noise) and you can gain more structural information on your analyte (QTOF) based on the fragmentation pattern.

I hope that helps

Cheers

Stefanie

Hi Mohammed,

Stefanie has given an excellent answer and adding to that, LC-MS/MS when used in MRM mode (multiple reaction monitoring mode) scanning for both precursor and product ion increases the specificity in addition to enhanced sensitivity. For example two compounds of same molecular weight (will produce same precursor ion) can be identified and quantified based on the different product ions formed after fragmentation. So this advantage exists when two mass analyzers work in tandem and because of this LC-MS/MS is also mentioned as tandem mass spectrometry.

Best regards,

Gopu

LC-MS is Liquid Chromatography (usually High Performance Liquid Chromatography, HPLC) combined with Mass Spectrometry.

 Here the sample in liquid form is injected into the LC and the different chemical components are separated (they travel at different speeds through the column due to differing affinities for the stationary phase, the coating on the inside of the column, and the mobile phase, the solvent passing through the column). The output of the LC column is then directed into a mass spectrometer where it is ionized (usually using electrospray ionization or atmospheric pressure chemical ionization) and a mass spectrum is generated. This allows better chemical identification or specificity.

Using LC MS-MS further increases the specificity. Typically a particular peak from the mass spectrum is selected and isolated and collisions are induced within the mass spectrometer to force a characteristic fragmentation of the selected ion. Because the mass spectrometer can perform these actions on timescales of seconds this can all happen while the LC is performing the initial chemical separation. As an example, structural isomers that would otherwise have a very similar chemical behaviour in the column and give the same ions in a single stage mass spectrum can give different fragment ions when an initial ion of a particular m/z is selected and fragmented.

I think Stefanie gave a good answer. There is just one thing to amend.

By pure physics, the sensiticvity of most instruments is higher (50x) in pure LC-MS mode. However, in most samples the sensitivity of LC MS is limited by the background of other compounds in the samples. MS/MS thus provides the selectivity to be able to determine trace amounts considering this MS/MS is usually something like (100x) more sensitive than MS. 

MS only measures the m/z of a compound. Whereas, MS/MS measures the m/z of the compound as well as its intermediates (by-products).

1. MS/MS filters the compound you target

2. then breaks it down into small intermediate compounds

3. then filters these intermediates and detect them

That provides extra precision and higher sensitivity

I agree with the explanation provided by Dr. Stefanie Kellner.

Regards

SM Najim

@ Arpith- Yes, it should work. As you noted, the peptide can take multiple charges. Just use the mass spectrometer to detect those multiple charges. The [M+3H]3+ ion will be at 1533 Da, within the range of the spectrometer that you have. I've detected peptides at ~7800 Da with a single quad MS with a limit of 2000 Da myself.

Clear with the answers. But when excision of proteins are done from SDS-PAGE, whether is there any difference in sample preparation and results analysis for LC-MS and LC-MS/MS? Particularly, I am working on virion particles from bacteriophages. 

 Will the LC-MS determine the mycotoxin Deoxinivalenol and its intermediates or do I have to use the LC-MS/MS?

In order to identify a molecule by LC-MS-MS, do you need a standard to compare the fragmentation pattern to?

Without going into the basic I will explain with a simple example . Consider that you have 2 compounds in a sample with similar molecular weight , means their m/z are same . In this case it would be difficult to quantify them separately . However , If you have two mass spec in tandem you can easily differentiate the compounds of similar molecular weight because two different compounds can have the similar mass but their daughter ions will not be the same.

Stefanie and Bester provided good answers to the differences between lc-ms and lc-ms/ms

Everyone is right, I would like to add; LC MS/MS has more sensitivity than LC MS.

Stephanie answear is perfect!

I agreed with Stefanie answer. Its like sieving a sand sample two times with different sieve aperture. The first one separates to a level and the second one gives a more refined particle size. i.e LC-MS/MS begins where LC-MS stops to give better information which LC-MS cannot give. Thanks.

MS measures mass:charge (ratio) of an ion, while MS/MS measures mass:charge as in MS followed by the mass:charge (second MS) of its constituent fragments. MS/MS offers higher sensitivity over MS.

Can anyone give more clear answer with simple example?

What types of sample are analysed by LC-MS-MS?

Muawuz Ijaz do you have any reference of your statement about the sensitivity of LCMS/MS

Harun, any type of sample can be analyzed by MS/MS as long as it can be fragmented to generate product ions. It increases specificity, sensitivity and confidence in the data.

Dear Sir,

We done our ESI-MS of our novel recombinant protein sample and found varying color (green,red,yellow) of peptide when matching to our known protein sequence (isolated and purified from plant source its ms spectra have identified 7 peptide and expressed it recombinantly). I have found all the peptide matching in our ESI-MS of recombinant protein with native protein ms (out of 7 peptide we have found 3 green 1 yellow and 3 red peptide). please clarify should i consider it my recombinant protein is have same sequence as native plant protein. Secondly what could be the reason of getting varying colour peptide.

Please clarify the significant of peptide deduced in sequence represented with varying color like green,Red and yellow. Could you please elaborate the percentage of similarity level of green, yellow and green peptides????

Thanks

Regards

I need a standard laboratory where I can send plant samples to for extraction and phtyhormone analysis using LC-MS/MS. Any suggestion will be appreciated.

Hi

1) In LC/MS, the effluent from the LC is ionized, and the ions are directed into a mass spectrometer. The spectrometer generally records all ions within the set range. You can collect all ions within the working range and plot the total ion chromatograph (TIC). If the analyte that you are observing has a unique mass, you can get better resolution of that analyte by performing SIM, single ion monitoring, where you only monitor the specific ion. You would use MS/MS if you need to confirm that the mass you are monitoring belongs to the analyte of interest. You could for instance monitor anisole at m/z 108. You would detect all ions at this m/z ratio, including other ions of other molecules such as benzyl alcohol, or fragments formed from degradation of larger molecules. You can perform MS/MS however. With the first mass separation, collect only m/z 108 ions. Then you would allow this ion to fragment and perform mass separation and look for a fragment with m/z 65 (C5H5+). That will give you further confirmation of the analyte's structure. 2)Some molecules form anions easier than cations, and vice versa. Sometimes you get more structural information using positive mode than negative mode, etc. Use the mode that is better suited for your analyte and your separation conditions.

Hi! I start to work with proteomic. And I`m having difficult to understand one information that is um my mzML file. What means the msLevel?

Thanks

Regards

Hello, dear maam. Do you mean programming or genome work?

Pamela C Lukasewicz Ferreira

msLevel gives you information on whether a scan is MS1 or MS2 or higher in case of multi stage mass spectrometry ( ion trap based workflows).

Sanjib Meitei Thanks for the help.

Regards

An LC-MS/MS is useful for fine-tuning the separation further and improving the resolution of ions segregated by single quadrupole instruments. Multiple reaction monitoring using LC-MS/MS offers many-fold selectivity improvements over single ion monitoring (SIM) using a single quadrupole LC-MS.

I hope it helps.

Regards.

Zeeshan

Hi Mohammad,

Go through this link to understand the MS technique.

https://www.ssi.shimadzu.com/sites/ssi.shimadzu.com/files/Products/literature/lcms/fundamental-guide-to-LCMS.pdf