Purification of tryptic peptides for mass spectrometry using polyvinylidene fluoride membrane.

A simple procedure for the purification of tryptic peptides, prior to mass spectrometric analysis, using polyvinylidene fluoride membrane (PVDF) is described. The sensitivity of mass spectrometric analysis is such that minor impurities in tryptic peptide digests suppress the signal obtained. However, we obtained useful signal, from a sample that did not yield any spectra earlier, by purifying the sample using PVDF membrane. For this, the tryptic peptide digest was first spotted on the membrane which was then air-dried and washed. Further, the membrane was extracted with trifluoroacetic acid (TFA) and acetonitrile and subjected to mass spectrometric analysis. This procedure enabled us to identify a cross-reactive D1 antigen on the neutrophil surface that bound antibodies that targeted 60 kD Ro autoantigen in systemic lupus erythematosus, an autoimmune disorder.

Heat mediated quick Coomassie blue protein staining and destaining of SDS-PAGE gels.

For the detection of proteins on sodium dodecyl sulphate-polyacrylamide gel electrophoresis, Coomassie blue is used commonly on account of its simplicity and reliability. In this report we show that enhanced heat, in addition to dramatically decreasing the time required for staining and destaining, also significantly increased the detection sensitivity. For a 1.5 mm gel, the staining time was 5 min at 55, 62.5 or 70 degrees C while the destaining time was 45, 45 and 20 min respectively. For a 0.8 mm gel, the staining time could be reduced to 1 min at 65 degrees C compared to 2 min at 60 degrees C and 5 min at 55 degrees C. The destaining time required was 8, 15 and 20 min at the respective temperatures.

Multiple nuclear dot antinuclear antibody in patients without primary biliary cirrhosis.

Multiple nuclear dot (MND), or pseudocentromere, anti-nuclear antibody (ANA) is an uncommon pattern associated primarily with primary biliary cirrhosis (PBC) and anti-mitochondrial antibody (AMA). A 53 kDa antigen with an apparent molecular mass of 100 kDa as found on sodium dodecyl sulphate-polyacrylamide gel electrophoresis is thought to be responsible for the uncommon pattern. This study analyzes sera from 21 patients without PBC or AMA that produced the uncommon MND ANA immunofluorescence pattern. Diseases present include lupus, rheumatoid arthritis and scleroderma. On immunoblotting nineteen of 21 (91%) bound a 70 kDa protein. Western blot analysis showed that this nuclear antigen was different from pyruvate dehydrogenase, p80 coilin and the antigen responsible for MND ANA in those with PBC. Affinity purified anti-70 kDa reproduced the MND ANA immunofluorescence pattern. Thus, the MND ANA in patients without PBC/AMA is associated with binding to a 70 kDa nuclear protein and not with a 53 kDa antigen (that runs at 100 kDa) found in those with MND and PBC/AMA. The data demonstrate the MND antigen without PBC/AMA is immunologically distinct from the pattern when found with PBC/AMA.