Barrie Gordon Wagenfeld, Age 7529 Lothrop St, Saugus, MA 01906

2003017, Sep 18, 2003

Dec 20, 2002

10/327342

Timothy Nadler - Framingham MA, US
Kenneth Parker - Hopkinton MA, US
George Vella - Medway MA, US
Barrie Wagenfeld - Saugus MA, US
Yulin Huang - Westwood MA, US
Robert Lotti - East Boston MA, US

C12Q001/37

435/023000

Polypeptides, for example those separated on a gel, are electroblotted through a digestion membrane to a composite capture membrane that can be directly analyzed using mass spectrometry. The molecular weights of the fragments generated by the digestion membrane are then used to identify the polypeptide from which they originated. The digestion membrane contains an immobilized protease such as trypsin, which cleaves the electroblotted proteins into fragments during electroblotting with such high enzyme cleavage capacity and efficiency that one pass of the polypeptide through the membrane is sufficient. The peptide fragments are collected onto a composite capture membrane that is chemically treated, for example, by adding a mixture of nitrocellulose and MALDI matrix, so as to absorb peptides near the surface to facilitate desportion, thereby increasing the sensitivity of subsequent analysis by MALDI-TOF mass spectrometry. In one application, labeling of proteins with reagents containing combinations of stable heavy isotopes prior to electroblotting and digestion permits the relative quantification of protein expression in two or more samples in a single operation. In another application, the gel containing separated polypeptides is replaced by a tissue sample, or blot of a tissue sample. The polypeptides from the tissue sample are then electroblotted through the digestion membrane onto the capture membrane, permitting the imaging of the tissue based on the proteins identified by mass spectrometry.