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Chromatography Forum Discuss Chromatography techniques in this forum. Includes a HPLC forum, and other "liquid phase" separation techniques.


iTRAQ and nano LC

iTRAQ and nano LC - Chromatography Forum

iTRAQ and nano LC - Discuss Chromatography techniques in this forum. Includes a HPLC forum, and other "liquid phase" separation techniques.


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Old 01-31-2011, 11:12 PM
Pipette Filler
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Default iTRAQ and nano LC



Hi to you all

I am doing proteomic analysis for leukimic B cells. First, Protein is extracted from cells then cleaned using 2D cleaning up kit. After that, protein extract is digested with trypsin. Then peptides are labeled with iTRAQ reagent and separated on LC (initially on SCX column then on RP column). Separated peptides are spotted on a MALDI plate to be analysed in MALDI TOF-TOF.

When I used unlabelled peptides (extracted from leukimic B cells) I was able achieve a good separation on the LC and get high number of identification in MALDI.

The problem has been that when peptides from the same sample were labeled with iTRAQ reagents, they were very poorly separated on the LC and thus low number of proteins was identified in MALDI.

I dont know what causing this poor separation for labeled peptides on The LC.

I wonder if anybody can help me to solve this problem.

Than you vey much in advance.

Suliman.
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