I have plasma samples and want to compare two groups to identify differentially expressed proteins by LC-MS. There are several approaches like label free and iTRAQ. I want to know which method is most suitable for comparison or quantification.
Narendra, plasma is difficult due to dynamic range, so you will probably need to employ sample fractionation steps to achieve analytical depth. Keep in mind that fractionation is more difficult (not impossible) to combine with label-free than with an early stage isotopic labeling. Another consideration might be the size of your groups - iTRAQ/TMT can be multiplexed, label-free is always parallel for instrument time. Look to the literature to see what other groups have been successful with.
You did not mentioned what insturment you have, ITRAQ is more optimized on applied biosystems MALDI. the other way is spectra counting. but you have to remove the high abundance proteins prior to get any data. we use the proteominer kit from biorad which does a good job,
Yes, you are right. I also used many kits from sigma as well as biorad's proteome minor. I have bruker's MALDI tof/tof (ultraflex) and amaZon speed ETD.
Following on Christof's comment, I would also recommend an isotope labelling type of approach, i.e. iTRAQ or TMT. Label-free studies, although well-regarded, require each sample to run separately. This is fine with an ESI LC-MS instrument but becomes quite laborious when using offline LC coupled to MALDI which is your situation. Data analysis also requires a bit of experience since no available software (commercial or freeware) is ideal. iTRAQ is more user friendly and would be a good start. iTRAQ ions can also be nicely detected in MALDI MS/MS so you shouldn't have a problem there. Good luck
Narendra: you'll be using the microtof and not the amazon? The microtof should work great with the iTRAQ, especially since it has nice low-m/z performance. I haven't evaluated the performance with fragment ions, but I know it's susceptible to a loss of performance in the low-m/z range of precursors when it gets dirty, so be careful to watch for that.
Already mentioned above, but it is better to repeat it.
Your first step is high abundant proteins removal (there are several options, mostly in the fashion of spin cartridge columns, that are able to remove up to 20 of the most abundant proteins).
Narendra, as mentioned above, the high abundance proteins in serum is the biggest problem, actually. I have been tried a kit called ProteinMiner from Bio-rad, which could be used for protein balance. It showed good results after using the kit. Then you could try label-free protocol for two groups quantification analysis. Good luck.