TY - JOUR
T1 - Top-down analysis of highly post-translationally modified peptides by Fourier transform ion cyclotron resonance mass spectrometry
AU - Guerrero, Andres
AU - Lerno, Larry
AU - Barile, Daniela
AU - Lebrilla, Carlito B
PY - 2015
Y1 - 2015
N2 - Bovine κ-caseinoglycomacropeptide (GMP) is a highly modified peptide from κ-casein produced during the cheese making process. The chemical nature of GMP makes analysis by traditional proteomic approaches difficult, as the peptide bears a strong net negative charge and a variety of post-translational modifications. In this work, we describe the use of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) for the top-down analysis of GMP. The method allows the simultaneous detection of different GMP forms that result from the combination of amino acid genetic variations and post-translational modifications, specifically phosphorylation and O-glycosylation. The different GMP forms were identified by high resolution mass spectrometry in both negative and positive mode and confirmation was achieved by tandem MS. The results showed the predominance of two genetic variants of GMP that occur as either mono- or bi-phosphorylated species. Additionally, these four forms can be modified with up to two O-glycans generally sialylated. The results demonstrate the presence of glycosylated, bi-phosphorylated forms of GMP never described before. [Figure not available: see fulltext.]
AB - Bovine κ-caseinoglycomacropeptide (GMP) is a highly modified peptide from κ-casein produced during the cheese making process. The chemical nature of GMP makes analysis by traditional proteomic approaches difficult, as the peptide bears a strong net negative charge and a variety of post-translational modifications. In this work, we describe the use of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) for the top-down analysis of GMP. The method allows the simultaneous detection of different GMP forms that result from the combination of amino acid genetic variations and post-translational modifications, specifically phosphorylation and O-glycosylation. The different GMP forms were identified by high resolution mass spectrometry in both negative and positive mode and confirmation was achieved by tandem MS. The results showed the predominance of two genetic variants of GMP that occur as either mono- or bi-phosphorylated species. Additionally, these four forms can be modified with up to two O-glycans generally sialylated. The results demonstrate the presence of glycosylated, bi-phosphorylated forms of GMP never described before. [Figure not available: see fulltext.]
KW - Caseinoglycomacropeptide
KW - Fourier transform ion cyclotron resonance mass spectrometry
KW - O-linked glycosylation
KW - Phosphorylation
KW - Top-down
UR - http://www.scopus.com/inward/record.url?scp=84923295873&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84923295873&partnerID=8YFLogxK
U2 - 10.1007/s13361-014-1034-5
DO - 10.1007/s13361-014-1034-5
M3 - Article
C2 - 25404158
AN - SCOPUS:84923295873
VL - 26
SP - 453
EP - 459
JO - Journal of the American Society for Mass Spectrometry
JF - Journal of the American Society for Mass Spectrometry
SN - 1044-0305
IS - 3
ER -