8 research outputs found
Comprehensive Profiling of N‑Linked Glycosylation Sites in HeLa Cells Using Hydrazide Enrichment
The adenocarcinoma cell line HeLa serves as a model
system for cancer research in general and cervical cancer in particular. In
this study, hydrazide enrichment in combination with state-of-the art
nanoLC−MS/MS analysis was used to profile N-linked glycosites in HeLa
cells. N-Linked glycoproteins were selectively enriched in HeLa cells by
the hydrazide capture method, which isolates all glycoproteins
independent of their glycans. Nonglycosylated proteins were removed
by extensive washing. N-Linked glycoproteins were identified with the
specific NXT/S motif and deamidated asparagine (N). Deglycosylation
was carried out in both H_2 (^16)O and H_2 ^(18)O to confirm the deamidation.
NanoLC−MS/MS analysis indicated that the method selectively enriched
at least 100 fold N-linked glycosites in HeLa cells. When both the
membrane and cytosolic fractions were used, a total of 268 unique N-glycosylation
sites were identified corresponding to 106 glycoproteins.
Bioinformatic analysis revealed that most of the glycoproteins identified
are known to have an impact on cancer and have been proposed as
biomarkers
Comprehensive profiling of N-linked glycosylation sites in HeLa cells using hydrazide enrichment
Comprehensive Profiling of N‑Linked Glycosylation Sites in HeLa Cells Using Hydrazide Enrichment
The adenocarcinoma cell line HeLa serves as a model system
for
cancer research in general and cervical cancer in particular. In this
study, hydrazide enrichment in combination with state-of-the art nanoLC–MS/MS
analysis was used to profile N-linked glycosites in HeLa cells. N-Linked
glycoproteins were selectively enriched in HeLa cells by the hydrazide
capture method, which isolates all glycoproteins independent of their
glycans. Nonglycosylated proteins were removed by extensive washing.
N-Linked glycoproteins were identified with the specific NXT/S motif
and deamidated asparagine (N). Deglycosylation was carried out in
both H<sub>2</sub><sup>16</sup>O and H<sub>2</sub><sup>18</sup>O to
confirm the deamidation. NanoLC–MS/MS analysis indicated that
the method selectively enriched at least 100 fold N-linked glycosites
in HeLa cells. When both the membrane and cytosolic fractions were
used, a total of 268 unique N-glycosylation sites were identified
corresponding to 106 glycoproteins. Bioinformatic analysis revealed
that most of the glycoproteins identified are known to have an impact
on cancer and have been proposed as biomarkers
Comprehensive Profiling of N‑Linked Glycosylation Sites in HeLa Cells Using Hydrazide Enrichment
The adenocarcinoma cell line HeLa serves as a model system
for
cancer research in general and cervical cancer in particular. In this
study, hydrazide enrichment in combination with state-of-the art nanoLC–MS/MS
analysis was used to profile N-linked glycosites in HeLa cells. N-Linked
glycoproteins were selectively enriched in HeLa cells by the hydrazide
capture method, which isolates all glycoproteins independent of their
glycans. Nonglycosylated proteins were removed by extensive washing.
N-Linked glycoproteins were identified with the specific NXT/S motif
and deamidated asparagine (N). Deglycosylation was carried out in
both H<sub>2</sub><sup>16</sup>O and H<sub>2</sub><sup>18</sup>O to
confirm the deamidation. NanoLC–MS/MS analysis indicated that
the method selectively enriched at least 100 fold N-linked glycosites
in HeLa cells. When both the membrane and cytosolic fractions were
used, a total of 268 unique N-glycosylation sites were identified
corresponding to 106 glycoproteins. Bioinformatic analysis revealed
that most of the glycoproteins identified are known to have an impact
on cancer and have been proposed as biomarkers
Comprehensive Profiling of N‑Linked Glycosylation Sites in HeLa Cells Using Hydrazide Enrichment
The adenocarcinoma cell line HeLa serves as a model system
for
cancer research in general and cervical cancer in particular. In this
study, hydrazide enrichment in combination with state-of-the art nanoLC–MS/MS
analysis was used to profile N-linked glycosites in HeLa cells. N-Linked
glycoproteins were selectively enriched in HeLa cells by the hydrazide
capture method, which isolates all glycoproteins independent of their
glycans. Nonglycosylated proteins were removed by extensive washing.
N-Linked glycoproteins were identified with the specific NXT/S motif
and deamidated asparagine (N). Deglycosylation was carried out in
both H<sub>2</sub><sup>16</sup>O and H<sub>2</sub><sup>18</sup>O to
confirm the deamidation. NanoLC–MS/MS analysis indicated that
the method selectively enriched at least 100 fold N-linked glycosites
in HeLa cells. When both the membrane and cytosolic fractions were
used, a total of 268 unique N-glycosylation sites were identified
corresponding to 106 glycoproteins. Bioinformatic analysis revealed
that most of the glycoproteins identified are known to have an impact
on cancer and have been proposed as biomarkers
Analysis of new growth promoting black market products
Detecting agents allegedly or evidently promoting growth such as human growth hormone (GH) or growth hormone releasing peptides (GHRP) in doping controls has represented a pressing issue for sports drug testing laboratories. While GH is a recombinant protein with a molecular weight of 22 kDa, the GHRPs are short (3-6 amino acids long) peptides with GH releasing properties. The endogenously produced GH (22 kDa isoform) consists of 191 amino acids and has a monoisotopic molecular mass of 22,124 Da. Within this study, a slightly modified form of GH was discovered consisting of 192 amino acids carrying an additional alanine at the N terminus, leading to a monoisotopic mass of 22,195 Da. This was confirmed by top-down and bottom-up experiments using liquid chromatography coupled to high resolution/high accuracy mass spectrometry. Additionally, three analogues of GHRPs were identified as Gly-GHRP-6, Gly-GHRP-2 and Gly-Ipamorelin, representing the corresponding GHRP extended by a N-terminal glycine residue. The structure of these peptides was characterised by means of high resolution (tandem) mass spectrometry, and for Gly-Ipamorelin and Gly-GHRP-2 their identity was additionally confirmed by custom synthesis. Further, established in-vitro experiments provided preliminary information considering the potential metabolism after administration