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Use of ZipTip Pipette Tips for Microscale Affinity Mass Spectrometry

bumin (BSA) was passed up and down the tip ten times to block the remaining unbound C18 sites. Following another 3 PBS washes, 20 μL of filtered human breast milk or pure transferrin at 0.01 mg/mL was repeatedly passed into and out of the ZipTip pipette tip for approximately 5 minutes. Following this incubation 2 PBS washes were performed followed by a wash with double-distilled water. To elute the bound transferrin, 3 μL of 1% TFA was passed up and down the tip ten times and finally eluted onto the MALDI plate and mixed with matrix or eluted into a buffered solution of trypsin at 20 μg/mL in 25 mM ammonium bicarbonate. If the sample was eluted into trypsin, it was incubated overnight at 37C. An aliquot was mixed with CHCA matrix and deposited on the MALDI plate.

Results and Discussion

After acid elution from the ZipTip pipette tip and MALDI-TOF mass spectrometry analysis, a peak at approximately 80 kDa was observed in the control experiment (pure transferrin) and the sample studied (breast milk) as shown in Figure 2, spectrum 1 and spectrum 3 respectively. Spectrum 2 represents the peaks observed after 50% MeCN elution from the ZipTip pipette tip, indicating that IgG was indeed bound to the tip. Spectrum 4 is human breast milk diluted 10-fold with sinapinic acid. Clearly, no transferrin was observed. Thus, specific binding of transferrin to the anti-transferrin ZipTip pipette tip was shown.

If the transferrin was eluted into trypsin and the resulting digest mass analyzed and data base searched, transferrin was clearly identified. Additional control experiments were performed to determine the specificity of the interaction between the transferrin and the anti-transferrin ZipTip pipette tip (Figure 3). These experiments were performed four times with similar results obtained each time To demonstrate that the interaction observed in Figure 2 was indeed specific and not just an interaction o
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