Serum protein profiling by miniaturized solid‐phase extraction and matrix‐assisted laser desorption/ionization mass spectrometry

Callesen, Anne Kjærgaard; Mohammed, Shabaz; Bunkenborg, Jakob; Kruse, Torben A.; Cold, Søren; Mogensen, Ole; Christensen, René dePont; Vach, Werner; Jørgensen, Per Schultz; Jensen, Ole N.

doi:10.1002/rcm.1960

Cited by 51 publications

(42 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The proteins were digested at 37°C overnight using trypsin (2 g enzyme/100 g protein), and the peptides were vacuum-dried to a volume of 20 l and acidified by adding formic acid. The tryptic peptides were desalted and concentrated on an Empore C18 extraction disk (3 M, St. Paul, MN) and reverse-phase POROS R3 (Perseptive Biosystems, Foster City, CA) packed in GELoader tips (Eppendorf, Hamburg, Germany) using a modified version of the previously described StageTips (14,21,22).…”

Section: Cell Culture and Stable Isotopic Labeling By Amino Acids In mentioning

confidence: 99%

NADH-Cytochrome b5 Reductase 3 Promotes Colonization and Metastasis Formation and Is a Prognostic Marker of Disease-Free and Overall Survival in Estrogen Receptor-Negative Breast Cancer*

Lund

Leth-Larsen

Caterino

et al. 2015

Molecular & Cellular Proteomics

Self Cite

View full text Add to dashboard Cite

Metastasis is the main cause of cancer-related deaths and remains the most significant challenge to management of the disease. Metastases are established through a complex multistep process involving intracellular signaling pathways. To gain insight to proteins central to specific steps in metastasis formation, we used a metastasis cell line model that allows investigation of extravasation and colonization of circulating cancer cells to lungs in mice. Using stable isotopic labeling by amino acids in cell culture and subcellular fractionation, the nuclear, cytosol, and mitochondria proteomes were analyzed by LC-MS/ MS, identifying a number of proteins that exhibited altered expression in isogenic metastatic versus nonmetastatic cancer cell lines, including NADH-cytochrome b5 reductase 3 (CYB5R3), L-lactate dehydrogenase A (LDHA), Niemann-pick c1 protein (NPC1), and nucleolar RNA helicase 2 (NRH2). The altered expression levels were validated at the protein and transcriptional levels, and analysis of breast cancer biopsies from two cohorts of patients demonstrated a significant correlation between high CYB5R3 expression and poor disease-free and overall survival in patients with estrogen receptor-negative tumors (DFS: p ‫؍‬ .02, OS: p ‫؍‬ .04). CYB5R3 gene knockdown using siRNA in metastasizing cells led to significantly decreased tumor burden in lungs when injected intravenously in immunodeficient mice. The cellular effects of CYB5R3 knock-down showed signaling alterations associated with extravasation, TGF␤ and HIF␣ pathways, and apoptosis. The decreased apoptosis of CYB5R3 knock-down metastatic cancer cell lines was confirmed in functional assays. Our study reveals a central role of CYB5R3 in extravasation/colonization of cancer cells and demonstrates the ability of our quantitative, comparative proteomic approach to identify key proteins of specific important biological processes that may also prove useful as potential biomarkers of clinical relevance.

show abstract

Section: Cell Culture and Stable Isotopic Labeling By Amino Acids In mentioning

confidence: 99%

NADH-Cytochrome b5 Reductase 3 Promotes Colonization and Metastasis Formation and Is a Prognostic Marker of Disease-Free and Overall Survival in Estrogen Receptor-Negative Breast Cancer*

Lund

Leth-Larsen

Caterino

et al. 2015

Molecular & Cellular Proteomics

Self Cite

View full text Add to dashboard Cite

show abstract

“…However, there have been concerns about the reproducibility and coverage of these techniques. MALDI-MS profiling of serum/ plasma proteins or peptides usually requires SPE pretreatment or pre-enrichment with functionalized beads or particles [59,60,[79][80][81][82]. Numerous studies have reported the use of SELDI-TOF-MS with different functional ProteinChips for disease detection mainly based on distinct proteomic patterns [83][84][85][86][87].…”

Section: Quantitative Analysis and Profilingmentioning

confidence: 99%

Human body fluid proteome analysis

2006

View full text Add to dashboard Cite

The focus of this article is to review the recent advances in proteome analysis of human body fluids, including plasma/serum, urine, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, synovial fluid, nipple aspirate fluid, tear fluid, and amniotic fluid, as well as its applications to human disease biomarker discovery. We aim to summarize the proteomics technologies currently used for global identification and quantification of body fluid proteins, and elaborate the putative biomarkers discovered for a variety of human diseases through human body fluid proteome (HBFP) analysis. Some critical concerns and perspectives in this emerging field are also discussed. With the advances made in proteomics technologies, the impact of HBFP analysis in the search for clinically relevant disease biomarkers would be realized in the future.

show abstract

“…For example, one study used solid-phase extraction and MALDI-TOF MS (25 ). In this study, 1 serum sample was prepared 8 times with C8 material and eluted directly onto the MALDI target.…”

Section: Reproducibility In Maldi Protein Profilingmentioning

confidence: 99%

Reproducibility in Protein Profiling by MALDI-TOF Mass Spectrometry

2007

View full text Add to dashboard Cite

Background: Protein profiling with high-throughput sample preparation and MALDI-TOF MS analysis is a new potential tool for diagnosis of human diseases. However, analytical reproducibility is a significant challenge in MALDI protein profiling. This minireview summarizes studies of reproducibility of MALDI protein profiling and current approaches to improve its analytical performance. Methods: The PubMed database was searched using combinations of the following search terms: MALDI, SELDI, reproducibility, variation, precision, peak intensity, quantification, peptide, biomarkers, and proteomics. Acceptance criteria were detailed reports on the reproducibility with MALDI protein profiling and studies describing efforts to improve the analytical performance with this technology. Results: The reported intraexperiment CVs of the peak intensity vary highly between individual protein peaks, with the reported mean CV of the peak intensity varying among studies from 4% to 26%. There is additional interexperiment variation in peak intensity. Current approaches to improve the analytical performance of MALDI protein profiling include automated sample processing, extensive prefractionation strategies, immunocapture, prestructured target surfaces, standardized matrix (co)crystallization, improved MALDI-TOF MS instrument components, internal standard peptides, quality-control samples, replicate measurements, and algorithms for normalization and peak detection. Conclusions: Further evaluation and optimization of MALDI-TOF MS is recommended before use in routine analysis.

show abstract

Serum protein profiling by miniaturized solid‐phase extraction and matrix‐assisted laser desorption/ionization mass spectrometry

Cited by 51 publications

References 29 publications

NADH-Cytochrome b5 Reductase 3 Promotes Colonization and Metastasis Formation and Is a Prognostic Marker of Disease-Free and Overall Survival in Estrogen Receptor-Negative Breast Cancer*

NADH-Cytochrome b5 Reductase 3 Promotes Colonization and Metastasis Formation and Is a Prognostic Marker of Disease-Free and Overall Survival in Estrogen Receptor-Negative Breast Cancer*

Human body fluid proteome analysis

Reproducibility in Protein Profiling by MALDI-TOF Mass Spectrometry

Contact Info

Product

Resources

About