PUNIAS (Protein Unfolding and Nano-Indentation Analysis Software) is a Graphical User Interface (GUI) high-throughput Atomic Force Microscope (AFM) force measurements analysis software.

PUNIAS is a user-friendly graphic user interface program enabling the analysis of a large amount of atomic force microscopy protein unfolding as well as nano-indentation curves data in very little time. The program generates all the data that can be obtained from the curves and analyses it, bypassing the need to analyze the data manually using a spreadsheet program.

PUNIAS can read data coming from Agilent Technologies, Veeco, Asylum Research, JPK, NT-MDT and Novascan facilities.

PUNIAS has first been developed for single-molecule force spectroscopy on a wide range of molecules. Such experiments not only give new insights into intra- and inter-molecular forces, they have also shown variations in physical parameters of individual molecules with respect to the mean values derived from ensemble measurements. But due to the stochastic nature of the forced unfolding experiments, a large number of experiments need to be acquired and analyzed in order to obtain accurate statistics (32,000 of such curves have been used in our paper: Carl et al. PNAS 98, 1565-1570, 2001).

To analyze such data obtained from an AFM facility, the files first needed to be exported into ASCII format. Then the data, which were in integer form, had to be multiplied by four parameters (hard and soft values) in order to express the extension and deflection values in nano meter. After this step, a curve could be plotted in a spreadsheet software (like Microsoft Excel), and from this curve the peaks and baseline were determined. Once all the data had been measured, it was then possible to calculate the unfolding length and force corresponding to the curve. This procedure was very repetitive and time consuming. Using this procedure it was possible to analyze 25 to 30 curves per day, depending on the number of unfolding peaks measured in the corresponding curves. It turns out that to analyze 1000 curves, a period of time of at least one month would have been needed.

Given this time frame, it was obvious to us from the moment we generated our first unfolding curves that it was necessary to develop a code or some method in order to treat a large amount of data in the minimum time possible. With the software we have written and are now using, it becomes possible to examine 1000 curves in about one hour.

Once PUNIAS had been developed for protein unfolding type of experiments on one AFM facility, it had been extended for nano-indentation type of experiments as well as for other facilities.