hello everyone and thank you for joining us for this video on how to prepare your samples for mass
spectrometry big shout out to everyone that joined that subscribed to this channel if you haven't yet
subscribed go ahead and hit the subscribe button and join the study group so in this video we're
going to look at how you prepare your samples for analysis via mass spectrometry so mass
spectrometry is to do with analyzing proteins or maybe you want to quantify proteins or maybe you
want to just characterize actually find out what kind of protein you are dealing with that's when
the technique of mass spectrometry is necessary it's one of several techniques but with this
technique you're able to identify proteins you're able to quantify proteins and you're also able to
characterize it look at any post-translational modifications that the protein may have so some
post-translational modifications like phosphorylation or glycosylation can be identified
by this technique to identify the proteins you first have to extract them you can extract them
from cells that you're culturing or you may so make a cell lysate or you may extract it from the
tissue which has the cells that are expressing the proteins that you want to characterize quantify or
identify the operation that you wish to identify or characterize or quantify is usually part of
a mixture of complex proteins other complex proteins so usually the strategy is to first
extract your protein via your reagent and physical method if necessary then you have a
step which is called reduction of the protein and then you have a step which is alkylation
and then you digest the protein with enzyme so it's trypsin but sometimes you have lysC also
as part of the mixture and then you clean up and the cleanup removes salts or detergents or other
components you use to prepare the proteins so that the sample is now clean enough to be ionized and
analyzed in a mass spectrometer in this video we'll focus on extracting proteins from tissues
this tends to require more complicated processing in the sense that you first
have to physically shear the tissue as lies the tissue as well as use reagent to lyse the
tissue where you are using just cell culture you just need to use reagent to lyse the cells
the lysis for mass spectrometry you need to keep in mind that detergents are a problem
ways that you can extract your proteins for lysis you can extract it with a kit such as the Pierce
kit or you can use what is called FASP stands for Filter Assisted Sample Preparation so use
a Filter Assisted Sample Preparation process. for in-solution digestion you usually denature
the proteins with chaotropic reagents such as urea before going on to sample reduction and alkylation
as you would whether you use the kit or the FASP process. so the reduction and alkylation steps are
to do with the cysteine amino acids cystine amino acids are reduced and then they are alkylated
to stop them from reforming the reduction in alkylations just allows you to irreversibly break
bonds between cysteine residues so they don't reform and the ultimate point of this is so you
don't get peptides that have been linked together by cysteine bonds which would make it hard for you
when you go to check what identify what protein it comes from because the databases wouldn't
have those kind of structures those kind of peptides that are linked via cysteines
sometimes you may want to just if you just want to identify one particular protein you may want
to run it on a gel and isolate that particular protein and then process it for mass spectrometric
analysis in order to characterize or quantify it. advantage of first running your protein through
a gel and then excising those bands another advantage is that it also provides a kind of
a cage for the proteins when you're processing so that when while you're doing your reduction
and your alkylations the proteins are encased and all caged inside the gel and it's only when
you trypsinize that the peptides are now small enough to release from the gel so it allows you
to wash out contaminants your your sodium dodecyl sulfate that you use to solubilize the protein and
run through the gel allows you to wash out salts that you've used in the processing and it's only
at the peptide stage that you're re that you now release your peptides for going ahead to acidify
uh for mass spectrometric analysis but if you are wanting to look at the complexity of proteins
under different conditions for instance you would you are likely to do an in-solution
digestion so you extract all the proteins so you use a detergent like sodium deoxycholate
which tends to be more compatible with mass spectrometry preparations and your chaotropic
reagent like urea then for really solubilizing everything you would sonicate a little bit
you would then centrifuge to just pellet out any debris that has not been fully homogenized and
solubilized and you want to do a quick protein concentration determination so you can just use
a Nanodrop and this step will just allow you to take a specific quantity of protein to go
and do your reduction or an alkylation of cysteine residues on they usually take about
a 100 microgram and then you go ahead and do the reduction and alkylation steps and such reagents
include TCEP or DTT, Tcep the full name is Tris (2 carboxyethyl) phosphine and DTT is Dithiothreitol
you reduce the cysteine residues then the free sulphur hydyl groups on the cysteine
residues are alkylated and you alkylate them with reagents such as iodoacetamide
or iodo-acetic acid and this irreversibly prevents the free sulfhydryls from reforming the disulfide
bonds after a reduction and alkylation steps in order for you to have the trypsin be active at
the required ph you usually need to resuspend the preparation in a larger volume to dilute out the
8 molar urea that you may have used or whatever concentration of lysis stronger concentration of
lysis buffer that you have used the other trips in the trypsin is usually either 1 in 150 or even
up to 1 in 100 so one part of your trypsin to 100 part of your proteins or 50 parts of the proteins
and you want to give it time to digest so you can digest it for several hours to overnight
to allow the trypsin to uh specifically cleave and fragment the proteins at particular residues
why do we use trypsin when we're digesting proteins for mass spectrometry if you know
the answer let me know in the comments following this step because you diluted
out the urea into a very large volume you want to now reduce the volume so that your protein is
concentrated so every injection of the protein into a mass spectrometer via liquid chromatography
column it has enough proteins to be analyzed so you want to reduce and usually you use a speed vac
where the liquids are evaporated of course with evaporation of the liquid you're now going to have
lots of the salts the Tris that you used or the other uh one molar urea that you still have in the
system is going to be very concentrated and this is why you need to create a solid face extraction
setup that allows you to extract out your peptides and elute out the salts and detergents so one
strategy is to create one yourself so just by using a 200 microliter tip you can go ahead
and put a column a disc that is composed of 18 carbons and then you can also include a cation
exchange disk having 18-carbons means that it will bind peptide but it will also be easy to
elute you know whereas if it's a protein it will bind really strongly and it won't you know because
there's lots of carbons in a protein versus a peptide because proteins more of the peptides
so then the proteins will not come off as easily but peptides will come off easily and then you're
basically going to load your sample on top of it and then centrifuge it and as you centrifuge it
all the salts and detergents will be eluted and your proteins will be bound by the clean up the
c18 tip be eluted into a collection tube so you want to go ahead and activate the
c18 carbon-18 discs that you're using to desalt so what you do is just add methanol you gently spin
this at 800 800 rcf for just about 30 seconds then you go ahead and add a second buffer
with this will be 85% Acetonitrile with 0.5 percent Trifluoroacetic acid (TFA) and you
spin this for five minutes and you're going to go ahead and remove any buffer that remains
lastly as part of the process of activating the stage tip you're going to add 50 microliters of
buffer that contains five percent ACN and point five percent TFA now you go ahead and acidify
your samples you acidify your samples by adding five percent ACN in point five percent TFA and
50 microliters of a 10% Trifluoroacetic acid (TFA) solution. You want a pH of less than four
so go ahead and test it with a pH strip acidified samples can now go on to the activated carbon-18
and cation exchange column that you've created in a 200 microliter pipette tip or a commercial one
that you've purchased first thing you do is to wash the sample that's now loaded onto the c18
column that you've created or purchased a wash is 50 microliters of 100 isopropanol with one
percent tfa second wash is performed at this time is with 50 microliters of 5 acn spiked with 0.2
percent Trifluoroacetic acid (TFA) you are now ready for elution pollute the peptides with an
elution buffer which consists of sixty percent acn and point five six percent ammonium hydroxide
diluted peptides it's in 50 microliters which is quite a large volume so you want to dry it down
and concentrate it and you do this by just putting it into centrifugal a centrifugal lyophilization
instrument such as the speedvac dry it down as much as possible without being completely bone dry
i suspend the lyophilized peptides in a slightly lower volume so about 20 microliters
or 15 microliters suspension at this point is 5% ACN with 0.5 percent Trifluoroacetic acid (TFA)
vortex it sonicate it for about five minutes then go ahead and check the
amount of peptides you have this is just very rough because you can't accurately quantify the
peptides this way but you get a rough estimate by using a nano drop at 280 nanometers for protein
quantitation and this first step for preparing to put into a mass spectrometer is that it's
gonna you're gonna inject about a microliter to five microliter depending on the concentration
once you have your acidified peptides then you're going to separate those peptides via
a high performance liquid chromatography column before it's injected into a mass spectrometer
via electrospray ionization (ESI) aim here is to get about a microgram of peptide per
injection then once inside the mass spectrometer you will obtain measurements that quantify how
much of each of the proteins that you have mass spec in itself is not inherently quantity based
just because of the different characteristics of the peptides so there are ways to get around it
so you can get quantities and then it will also do and what is called an ms2 to identify the peptide
so the ms-1 gives you the quantities the initial fragmentations to give you the quantities and
the ms2 the mass spec 2 will go ahead and identify the peptides so the identification of the peptides
that spectra you submit to dedicated databases or softwares algorithms that allows you to
work out what those peptides that spectra relates to in terms of what peptide will give you that
spectra and some of those dedicated algorithms includes max quant and then you go ahead and
identify the protein from based on the peptides that have been identified and that's when you
would use database like uniprot so at this point what you should also be aware of is that you've
alkylated all the cysteines in your peptides so during your software searches of your spectra
you will need to select the option that says that all your systems have been alkylated
and the reason here is that you've changed the native mass of the cysteines by alkylating it
so it needs to adjust for a heavier cysteine than what would be native
some instances you may be interested in post-translationally modified (PTMs) proteins
or particular peptides and if you are then you will do what is called an enrichment
step an enrichment step just means of all the proteins that you've extracted
you will focus on the ones that you're actually interested in and use reagents or uh setups
that allow you to enrich for those to get lots of those to analyze rather than just putting
all the complex mixture into the mass spectrometer once you release proteins into solution you know
there are proteases that will start to degrade proteins so it is recommended if your protocol
you may need to put in protease inhibitors as part of the process
so that's it for how to conduct in-solution digestion of tissue samples for mass spectrometry
using urea using a chaotropic reagent like urea so i'm going to leave it here and let
me know if you want us to look at a mass spectra together in another video okay bye bye God bless