6 research outputs found
MOESM1 of Antigen 85B peptidomic analysis allows species-specific mycobacterial identification
Additional file 1: Table S1. Ag85B target peptide identification and quantification by LC-MS/MS PRM mode
Profiling and Relative Quantitation of Phosphoinositides by Multiple Precursor Ion Scanning Based on Phosphate Methylation and Isotopic Labeling
Phosphoinositides,
the phosphorylated derivatives of phosphatidylinositol
(PtdIns), are key regulators of many fundamental biological processes,
including cell growth, proliferation, and motility. Here, we present
a novel method for rapid, sensitive, and simultaneous profiling of
phosphatidylinositol trisphosphate (PtdInsP<sub>3</sub>), phosphatidylinositol
bisphosphate (PtdInsP<sub>2</sub>), and phosphatidylinositol phosphate
(PtdInsP) of different fatty acid compositions. This method is based
on a technique called “charged diacylglycerol fragment ion-specific
multiple precursor ion scanning” (DAG<sup>+</sup>-specific
MPIS), coupled with prior phosphate methylation. Using DAG<sup>+</sup>-specific MPIS, we were able to identify 32 PtdIns, 28 PtdInsP, 30
PtdInsP<sub>2</sub>, and 3 PtdInsP<sub>3</sub> molecular species from
bovine brain extracts or prostatic cancer cell lines in an efficient
and time-saving manner. Our analysis revealed a large range of fatty
acyl compositions in phosphoinositides not obtained previously from
mammalian samples. We also developed a method that involves isotopic
labeling of endogenous phosphoinositides with deuterated diazomethane
(CD<sub>2</sub>N<sub>2</sub>) for quantitation of phosphoinositides.
CD<sub>2</sub>N<sub>2</sub> was generated in situ through acid-catalyzed
H/D exchange and methanolysis of trimethylsilyl diazomethane (TMS-diazomethane).
Phosphoinositides, extracted from a PC3 prostatic cancer cell line,
were labeled either with CH<sub>2</sub>N<sub>2</sub> or CD<sub>2</sub>N<sub>2</sub> and mixed in known proportions for DAG<sup>+</sup>-specific
MPIS-based mass spectrometry (MS) analysis. The results indicate that
isotopic labeling is capable of providing accurate quantitation of
PtdInsP<sub>3</sub>, PtdInsP<sub>2</sub>, and PtdInsP with adequate
linearity as well as high reproducibility with an average coefficient
variation of 18.9%. More importantly, this new methods excluded the
need for multiple phosphoinositide internal standards. DAG<sup>+</sup>-specific MPIS and isotopic labeling based MS analysis of phosphoinositides
offers unique advantages over existing approaches and presents a powerful
tool for research of phosphoinositide metabolism
Evaluation of Different N‑Glycopeptide Enrichment Methods for N‑Glycosylation Sites Mapping in Mouse Brain
N-Glycosylation of
proteins plays a critical role in many biological
pathways. Because highly heterogeneous N-glycopeptides are present
in biological sources, the enrichment procedure is a crucial step
for mass spectrometry analysis. Five enrichment methods, including
IP-ZIC-HILIC, hydrazide chemistry, lectin affinity, ZIC-HILIC-FA,
and TiO<sub>2</sub> affinity were evaluated and compared in the study
of mapping N-glycosylation sites in mouse brain. On the basis of our
results, the identified N-glycosylation sites were 1891, 1241, 891,
869, and 710 and the FDR values were 3.29, 5.62, 9.54, 9.54, and 20.02%,
respectively. Therefore, IP-ZIC-HILIC enrichment method displayed
the highest sensitivity and specificity. In this work, we identified
a total of 3446 unique glycosylation sites conforming to the N-glycosylation
consensus motif (N-X-T/S/C; X ≠P) with <sup>18</sup>O labeling
in 1597 N-glycoproteins. N-glycosylation site information was used
to confirm or correct the transmembrane topology of the 57 novel transmembrane
N-glycoproteins
Proteomic Comparison and MRM-Based Comparative Analysis of Metabolites Reveal Metabolic Shift in Human Prostate Cancer Cell Lines
One of the major
challenges in prostate cancer therapy remains
the development of effective treatments for castration-resistant prostate
cancer (CRPC), as the underlying mechanisms for its progression remain
elusive. Previous studies showed that androgen receptor (AR) is crucially
involved in regulation of metabolism in prostate cancer (PCa) cells
throughout the transition from early stage, androgen-sensitive PCa
to androgen-independent CRPC. AR achieves such metabolic rewiring
directively either via its transcriptional activity or via interactions
with AMP-activated protein kinase (AMPK). However, due to the heterogeneous
expression and activity status of AR in PCa cells, it remains a challenge
to investigate the links between AR status and metabolic alterations.
To this end, we compared the proteomes of three pairs of androgen-sensitive
(AS) and androgen-independent (AI) PCa cell lines, namely, PC3-AR<sup>+</sup>/PC3, 22Rv1/Du145, and LNCaP/C42B, using an iTRAQ labeling
approach. Our results revealed that most of the differentially expressed
proteins between each pair function in metabolism, indicating a metabolic
shift between AS and AI cells, as further validated by multiple reaction
monitoring (MRM)-based quantification of nucleotides and relative
comparison of fatty acids between these cell lines. Furthermore, increased
adenylate kinase isoenzyme 1 (AK1) in AS relative to AI cells may
result in activation of AMPK, representing a major regulatory factor
involved in the observed metabolic shift in PCa cells
Proteomic Comparison and MRM-Based Comparative Analysis of Metabolites Reveal Metabolic Shift in Human Prostate Cancer Cell Lines
One of the major
challenges in prostate cancer therapy remains
the development of effective treatments for castration-resistant prostate
cancer (CRPC), as the underlying mechanisms for its progression remain
elusive. Previous studies showed that androgen receptor (AR) is crucially
involved in regulation of metabolism in prostate cancer (PCa) cells
throughout the transition from early stage, androgen-sensitive PCa
to androgen-independent CRPC. AR achieves such metabolic rewiring
directively either via its transcriptional activity or via interactions
with AMP-activated protein kinase (AMPK). However, due to the heterogeneous
expression and activity status of AR in PCa cells, it remains a challenge
to investigate the links between AR status and metabolic alterations.
To this end, we compared the proteomes of three pairs of androgen-sensitive
(AS) and androgen-independent (AI) PCa cell lines, namely, PC3-AR<sup>+</sup>/PC3, 22Rv1/Du145, and LNCaP/C42B, using an iTRAQ labeling
approach. Our results revealed that most of the differentially expressed
proteins between each pair function in metabolism, indicating a metabolic
shift between AS and AI cells, as further validated by multiple reaction
monitoring (MRM)-based quantification of nucleotides and relative
comparison of fatty acids between these cell lines. Furthermore, increased
adenylate kinase isoenzyme 1 (AK1) in AS relative to AI cells may
result in activation of AMPK, representing a major regulatory factor
involved in the observed metabolic shift in PCa cells
Proteomic Comparison and MRM-Based Comparative Analysis of Metabolites Reveal Metabolic Shift in Human Prostate Cancer Cell Lines
One of the major
challenges in prostate cancer therapy remains
the development of effective treatments for castration-resistant prostate
cancer (CRPC), as the underlying mechanisms for its progression remain
elusive. Previous studies showed that androgen receptor (AR) is crucially
involved in regulation of metabolism in prostate cancer (PCa) cells
throughout the transition from early stage, androgen-sensitive PCa
to androgen-independent CRPC. AR achieves such metabolic rewiring
directively either via its transcriptional activity or via interactions
with AMP-activated protein kinase (AMPK). However, due to the heterogeneous
expression and activity status of AR in PCa cells, it remains a challenge
to investigate the links between AR status and metabolic alterations.
To this end, we compared the proteomes of three pairs of androgen-sensitive
(AS) and androgen-independent (AI) PCa cell lines, namely, PC3-AR<sup>+</sup>/PC3, 22Rv1/Du145, and LNCaP/C42B, using an iTRAQ labeling
approach. Our results revealed that most of the differentially expressed
proteins between each pair function in metabolism, indicating a metabolic
shift between AS and AI cells, as further validated by multiple reaction
monitoring (MRM)-based quantification of nucleotides and relative
comparison of fatty acids between these cell lines. Furthermore, increased
adenylate kinase isoenzyme 1 (AK1) in AS relative to AI cells may
result in activation of AMPK, representing a major regulatory factor
involved in the observed metabolic shift in PCa cells