Hello,
I am new to the field of proteomics and interested in analysing aa composition of protein product (mainly storage protein). Have access to aa analyser but due to high running cost I'd rather use an alternative method for an early exploration.
From literature I found that MS using techniques are quite common, however there's a whole array of different setups (GC - MS, LC - MS, CE - MS, LC- MS/MS etc.). My main questions are which setup would be most suitable for protein AAA and free AAA at this stage I am more interested in lower cost, reproducibility and decent throughput rather than very high sensitivity. I don't have any experience with working with these pieces of equipment so I find it difficult to find a good starting point for my analyses. I will be very grateful for any advice on the matter.
Many thanks
Kamil
GC-MS will have little or no utility for protein analysis because proteins are not volatile. LC-MS is useful, but suffers from being less specific than is often needed. (This is not always true, but is often the case.) CE-MS can often have greater specificity than LC-MS, but CE-MS has historically been more troublesome to use than LC-MS. This may or may not be true at present.
Your best bet is LC-MS/MS, if you can afford the cost. You can run those instruments as either LC-MS or LC-MS/MS. Regarding the MS/MS part, your choice of technology will be dictated by your application needs. The most common choice is triple quadrupole. However, other choices for the MS/MS part have other tradeoffs between advantages and disadvantages. For example, a Q-TOF has relatively high resolution for product ions, but the sensitivity for any specific product ion is less than it is when using a triple quadrupole. On the other hand, if you want to acquire a full product ion spectrum then the Q-TOF tends to be more sensitive than a triple quadrupole.
GC-MS will have little or no utility for protein analysis because proteins are not volatile. LC-MS is useful, but suffers from being less specific than is often needed. (This is not always true, but is often the case.) CE-MS can often have greater specificity than LC-MS, but CE-MS has historically been more troublesome to use than LC-MS. This may or may not be true at present.
Your best bet is LC-MS/MS, if you can afford the cost. You can run those instruments as either LC-MS or LC-MS/MS. Regarding the MS/MS part, your choice of technology will be dictated by your application needs. The most common choice is triple quadrupole. However, other choices for the MS/MS part have other tradeoffs between advantages and disadvantages. For example, a Q-TOF has relatively high resolution for product ions, but the sensitivity for any specific product ion is less than it is when using a triple quadrupole. On the other hand, if you want to acquire a full product ion spectrum then the Q-TOF tends to be more sensitive than a triple quadrupole.
Hi Kamil,
As nicely put forth above, it's got to be LC-MS/MS ! It is very standardized and easy for the field.
Thanks,
John R Carney Thank you for the suggestion. I will have to look into that. I am interested in comparing effect of different media/manipulations of AA synthesis pathways on AA composition of storage protein. I will also be looking at free AAs levels to get the full picture but my main point of focus is protein composition.
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