Protein 1 standard calibration mixture

A 10 μl protein sample was desalted using ZipTip^® C4 micro-columns (Merck Millipore, Burlington, MS, USA) and eluted with 0.5 μl SA [sinapinic acid, 10 mg/ml in [70:30] Acetonitrile: Trifluoroacetic acid 0.1%] matrix onto a GroundSteel massive 384 target (Bruker Daltonics, Billerica, MA, USA). An Autoflex III MALDI-TOF/TOF spectrometer (Bruker Daltonics) was used in linear mode with the following settings: 5,000–40,000 Th window, linear positive mode, ion source 1: 20 kV, ion source 2: 18.5 kV, lens: 9 kV, pulsed ion extraction of 120 ns, high gating ion suppression up to 1,000 Mr. Mass calibration was performed externally with protein 1 standard calibration mixture (Bruker Daltonics). Data acquisition, peak peaking and subsequent spectra analysis was performed using FlexControl 3.0 and FlexAnalysis 3.0 software (Bruker Daltonics).

A 2 μl protein sample was desalted using ZipTip® C4 micro-columns (Merck Millipore) and eluted with 0.5 μl sinapinic acid (10 mg/ml in [70:30] Acetonitrile: Trifluoroacetic acid 0.1%) matrix onto a GroundSteel massive 384 target (Bruker Daltonics). An Autoflex III MALDI-TOF/TOF spectrometer (Bruker Daltonics) was used in linear mode with the following settings: 5000–40,000 Th window, linear positive mode, ion source 1: 20 kV, ion source 2: 18.5 kV, lens: 9 kV, pulsed ion extraction of 120 ns, high gating ion suppression up to 1000 Mr. Mass calibration was performed externally with protein 1 standard calibration mixture (Bruker Daltonics). Data acquisition, peak peaking, and subsequent spectra analysis was performed using the FlexControl 3.0 and FlexAnalysis 3.0 software (Bruker Daltonics).

The stable fragment of Sap1 obtained following thermolysin treatment was purified by reverse phase HPLC on a ZORBAX 300SB C18 column (Agilent). The molecular mass of the Sap1 stable fragment was determined by MALDI-TOF mass spectrometry. For MALDI-TOF the sample was spotted using sinapinic acid [10 mg/ml] in [70:30] acetonitrile-trifluoroacetic acid [0.1%]) on a MTP 384 target plate (Bruker Daltonics). An Autoflex III MALDI-TOF/TOF spectrometer (Bruker Daltonics) was used in linear mode with the following settings: 5,000- to 50,000-Da window; linear positive mode; ion source 1, 20 kV; ion source 2, 18.5 kV; lens, 9 kV; pulsed ion extraction of 120 ns; and high gating ion suppression up to 1,000 M_r. Mass calibration was performed externally with Bruker’s Protein 1 standard calibration mixture (Bruker Daltonics). Data acquisition was performed using the FlexControl 3.0 software program (Bruker Daltonics), and peak searching and subsequent spectral analysis were performed using FlexAnalysis 3.0 software (Bruker Daltonics).