Preventing carryover of peptides and proteins in nano LC-MS separations.
Sample carryover is a significant problem that occurs in high-performance liquid chromatography (HPLC) analysis. Carryover effects cannot be tolerated in any high-performance liquid chromatography-mass spectroscopy (HPLC-MS) separation system, and proteomics analysis must be performed in a separation system with virtually no carryover. Several procedures have been tested for effective and fast removal of interfering peptides and proteins originating from previous analyses in the HPLC system. We have developed and optimized a cleaning method for eliminating carryover caused by the autosampler and the trap column. The new washing method uses an injection of trifluoroethanol into the injection path and onto the trap column to remove strongly bound peptides and proteins, and it includes trifluoroethanol as an additional solvent in the chromatographic mobile phase for enhanced cleaning of the separation column. By application of this method, a significant reduction in carryover was achieved without any loss in the amount of proteins and peptides identified by MS.
Mitulović
Goran
G
Institute of Molecular Biotechnology of the Austrian Academy of Science, Dr. Bohr-Gasse 3, A-1030 Vienna, Austria. mitulovic@imp.ac.at
Stingl
Christoph
C
Steinmacher
Ines
I
Hudecz
Otto
O
Hutchins
James R A
JR
Peters
Jan-Michael
JM
Mechtler
Karl
K
D000595:Amino Acid Sequence; D000818:Animals; D016477:Artifacts; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007267:Injections; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D036103:Nanotechnology; D010455:Peptides / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results
D010455:Peptides; D011506:Proteins