MALDI-TOF High Mass Calibration up to 200 kDa Using Human Recombinant 16 kDa Protein Histidine Phosphatase Aggregates

摘要

Protein histidine phosphatase (PHP) hydrolytically cleaves phosphate groups from histidine residues. It was described for vertebrates in the year 2002 [1, 2]. ATP-citrate lyase [3] and other substances have been identified as physiological substrates. Recombinantly expressed human 16 kDa PHP [4] forms multimeric complexes in physiological buffer [5]. Regular series of PHP multimers can also be observed up to 200 kDa in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Since high-quality PHP can be produced, its use as calibration compound for high mass ranges is discussed. Available calibration standards typically cover the range up to 100 kDa and are based on well known proteins such as apomyoglobin, trypsinogen and bovine serum albumin of high to moderate purity. Calibration of higher mass ranges is still not an easy task. While proteins of larger molecular weight can be purchased, e.g. standards for gel electrophoresis, they are often mircoheterogeneous and their purity is mostly not sufficient for MS measurement resulting in peaks of low resolution. Authors have suggested use of different classes of aggregate or macromolecule forming substances such as poly(dimethylsiloxanes) [6], but those compounds are only available as mixtures of average molecular weight so that their use as calibrants is limited to very specific applications.