Depletion of human serum albumin in embryo culture media for in vitro fertilization using monolithic columns with immobilized antibodies.
Affinity depletion of abundant proteins such as HSA is an important stage in routine sample preparation prior to MS/MS analysis of biological samples with high range of concentrations. Due to the charge competition effects in electrospray ion source that results in discrimination of the low-abundance species, as well as limited dynamic range of MS/MS, restricted typically by three orders of magnitude, the identification of low-abundance proteins becomes a challenge unless the sample is depleted from high-concentration compounds. This dictates a need for developing efficient separation technologies allowing fast and automated protein depletion. In this study, we performed evaluation of a novel immunoaffinity-based Convective Interaction Media analytical columns (CIMac) depletion column with specificity to HSA (CIMac-αHSA). Because of the convective flow-through channels, the polymethacrylate CIMac monoliths afford flow rate independent binding capacity and resolution that results in relatively short analysis time compared with traditional chromatographic supports. Seppro IgY14 depletion kit was used as a benchmark to control the results of depletion. Bottom-up proteomic approach followed by label-free quantitation using normalized spectral indexes were employed for protein quantification in G1/G2 and cleavage/blastocyst in vitro fertilization culture media widely utilized in clinics for embryo growth in vitro. The results revealed approximately equal HSA level of 100 ± 25% in albumin-enriched fractions relative to the nondepleted samples for both CIMac-αHSA column and Seppro kit. In the albumin-free fractions concentrated 5.5-fold by volume, serum albumin was identified at the levels of 5-30% and 20-30% for the CIMac-αHSA and Seppro IgY14 spin columns, respectively.
V.L. Talrose Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences, Moscow, Russia.
Irina A
IA
Tarasova
V.L. Talrose Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences, Moscow, Russia.|Moscow Institute of Physics and Technology (State University), Dolgoprudny, Moscow, Russia.
Anna A
AA
Lobas
BIA Separations d.o.o, Ajdovščina, Slovenia.
Urh
U
Černigoj
V.L. Talrose Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences, Moscow, Russia.|Moscow Institute of Physics and Technology (State University), Dolgoprudny, Moscow, Russia.
Elizaveta M
EM
Solovyeva
Clinical Instititute of Laboratory Medicine, Medical University of Vienna, Vienna, Austria.
Barbara
B
Mahlberg
V.L. Talrose Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences, Moscow, Russia.|Moscow Institute of Physics and Technology (State University), Dolgoprudny, Moscow, Russia.
Mark V
MV
Ivanov
Clinical Instititute of Laboratory Medicine, Medical University of Vienna, Vienna, Austria.
Tanja
T
Panić-Janković
Faculty of Science and Technology, University of Debrecen, Debrecen, Hungary.
Zoltan
Z
Nagy
V.L. Talrose Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences, Moscow, Russia.
Marina L
ML
Pridatchenko
Faculty of Science and Technology, University of Debrecen, Debrecen, Hungary.
Andras
A
Pungor
BIA Separations d.o.o, Ajdovščina, Slovenia.
Blaž
B
Nemec
BIA Separations d.o.o, Ajdovščina, Slovenia.
Urška
U
Vidic
BIA Separations d.o.o, Ajdovščina, Slovenia.
Jernej
J
Gašperšič
BIA Separations d.o.o, Ajdovščina, Slovenia.
Nika Lendero
NL
Krajnc
BIA Separations d.o.o, Ajdovščina, Slovenia.
Jana
J
Vidič
V.L. Talrose Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences, Moscow, Russia.|Moscow Institute of Physics and Technology (State University), Dolgoprudny, Moscow, Russia.
Mikhail V
MV
Gorshkov
Clinical Instititute of Laboratory Medicine, Medical University of Vienna, Vienna, Austria. goran.mitulovic@meduniwien.ac.at.
Goran
G
Mitulović
D003470:Culture Media; D004622:Embryo, Mammalian; D005307:Fertilization in Vitro; D006801:Humans; D012709:Serum Albumin / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
D003470:Culture Media; D012709:Serum Albumin
Convective interaction media (CIM) chromatographic monoliths; Immobilized antibodies; LC-MS/MS sample preparation; Protein depletion