MALDI linear TOF mass spectrometry of PEGylated (glyco)proteins.
PEGylation of proteins is a fast growing field in biotechnology and pharmaceutical sciences owing to its ability to prolong the serum half-life time of recombinant proteins. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) has been shown to be a powerful tool in the analysis of several PEGylated small proteins. Here we present data obtained with a standard secondary electron multiplier (SEM) and a high mass (HM) detector combined with a MALDI linear TOF MS system for the detection of PEGylated (glyco)proteins in the range of 60-600 kDa. Examples of MALDI TOF MS of small (interferon alpha2a), middle (human serum albumin (HSA)) and high molecular mass proteins (coagulation factor VIII and von Willebrand factor (vWF), both heavily glycosylated proteins) are presented. The particular challenge for the analysis was the heterogeneity of the (glyco)proteins in the high molecular weight range in combination with additional PEGylation, which even introduced more heterogeneity and was more challenging for interpretation. Nevertheless, the performance of MALDI linear TOF MS with both detector systems in terms molecular weight and heterogeneity determination depending on the m/z range was superior to the other methods. Although the SEM was able to obtain information about protein PEGylation in the mass range up to 100 kDa (e.g. PEGylated HSA), the HM system was crucial for detection of HM ions (e.g. PEGylated recombinant vWF), which was impossible with the standard SEM.
Seyfried
Birgit K
BK
Institute of Chemical Technologies and Analytics, Vienna University of Technology, Vienna, Austria.
Siekmann
Jürgen
J
Belgacem
Omar
O
Wenzel
Ryan J
RJ
Turecek
Peter L
PL
Allmaier
Günter
G
D005169:Factor VIII / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry*; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry; D008970:Molecular Weight; D011092:Polyethylene Glycols / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012709:Serum Albumin / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014841:von Willebrand Factor / Q000737:chemistry
D006023:Glycoproteins; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins; D012709:Serum Albumin; D014841:von Willebrand Factor; D011092:Polyethylene Glycols; C078147:F8 protein, human; D005169:Factor VIII; C100416:peginterferon alfa-2a