A new fast method for nanoLC-MALDI-TOF/TOF-MS analysis using monolithic columns for peptide preconcentration and separation in proteomic studies.
A new fast method for identification and characterization of proteolytic digests of proteins by monolithic liquid chromatography coupled with mass spectrometry has been developed. The advantages of the monolithic columns are a high-pressure stability and low back pressure resulting in higher flow rates for capillary or nanosize columns simplifying the system handling. As was shown in several publications, such monolithic stationary phases are highly qualified for the analysis of peptides and proteins, but so far, only small volumes could be injected into the system, which might hamper the sample preparation leading to protein precipitation and partial loss of sample. To overcome the problem of small injection volumes, we established a system including a short monolithic trap column to allow preconcentration of the peptides. The injected sample is flushed at higher flow rates onto the trap column, bound to the stationary phase, and in this way concentrated in a few nanoliters before starting the separation. The expanded system was optimized and tested using different reference protein samples. Eluting peptides were detected by MALDI-TOF/TOF-MS and identified by database searching. The system is now a permanent part for proteome analysis in our lab, and as such, it was successfully applied for the detection of post-translational modifications and the analysis of membrane proteins. One example for these analyses is also included in this paper.
Marcus
Katrin
K
Medizinisches Proteom-Center, Ruhr-University Bochum, Universitätstrasse 150, 44780 Bochum, Germany. katrin.marcus@rub.de
Schäfer
Heike
H
Klaus
Stefan
S
Bunse
Christian
C
Swart
Remco
R
Meyer
Helmut E
HE
D000595:Amino Acid Sequence; D045304:Cytochromes c / Q000737:chemistry / Q000302:isolation & purification; D005949:Glucose Oxidase / Q000737:chemistry / Q000302:isolation & purification; D008049:Lipase / Q000737:chemistry / Q000302:isolation & purification; D008969:Molecular Sequence Data; D053758:Nanoparticles; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification*; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D038202:alpha-Crystallin A Chain / Q000737:chemistry / Q000302:isolation & purification
D010455:Peptides; D038202:alpha-Crystallin A Chain; D045304:Cytochromes c; D005949:Glucose Oxidase; D008049:Lipase