Nanoliter sample handling combined with microspot MALDI-MS for detection of gel-separated phosphoproteins.
We describe a microspot matrix-assisted laser desorption ionization (MALDI) mass spectrometric approach to analyze gel-separated phosphoproteins. This method involves in-gel digestion of phosphoproteins after gel separation, followed by open tubular capillary (OTC) immobilized metal-ion affinity chromatography (IMAC) to capture the phosphopeptides with markedly reduced interferences from nonphosphorylated peptides. Nanoliter-volume of ammonium phosphate is used to elute the phosphopeptides captured on the capillary tube. After mixing with a small volume of matrix solution in the capillary, the effluent is deposited in a microspot on a sample plate for MALDI-MS analysis. It is also shown that, with peptide esterification after in-gel digestion of a phosphoprotein, negative ion detection in MALDI gives a distinct advantage over the positive ion mode of operation for phosphopeptide analysis, even without IMAC enrichment. However, the OTC-IMAC technique is demonstrated to be superior to the approach of negative ion detection of esterified in-gel digests without IMAC. OTC-IMAC is found to be sufficiently selective to capture phosphopeptides from in-gel digest of a gel band containing predominately one protein and the combination of peptide esterification and IMAC enrichment does not provide any real advantage. Using a standard phosphoprotein alpha-casein as a model system, we demonstrate that this OTC-IMAC method can detect a number of phosphopeptides after in-gel digestion with mid-fmol protein sample loading. An example of real world applications of this method is illustrated in the characterization of a fusion protein, His182, expressed in E. coli.
Stupak
Jacek
J
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
Liu
Huaizhi
H
Wang
Zhengping
Z
Brix
Brenda J
BJ
Fliegel
Larry
L
Li
Liang
L
D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D036103:Nanotechnology; D010750:Phosphoproteins / Q000032:analysis* / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D010750:Phosphoproteins