Quantification of Serum Proteins of Metastatic Oral Cancer Patients Using LC-MS/MS and iTRAQ Labeling

Abstract

Metastasis is a critical event in oral squamous cell carcinoma (OSCC) progression. In this study, we have performed quantitative analysis of serum proteins from non-metastatic (lymph-node metastasis free) and metastatic OSCC patients using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with iTRAQ labeling (isobaric tagging for relative and absolute quantitation). To eliminate highly abundant proteins, the serum samples were initially separated by SDS-PAGE and only low abundant protein bands were excised for subsequent in-gel tryptic digestion. The resulting peptides were then extracted from each sample gels and labeled with iTRAQ reagent 114 (control), 116 (non-metastatic) and 117 (metastatic), respectively. Afterwards, the labeled samples were combined and subjected to LC-MS/MS analysis using linear ion trap (LIT) MS with pulsed Q collision induced dissociation (PQD). A total of 64 proteins were identified and quantified by this approach. Our study showed that iTRAQ labeling and LIT-MS with PQD is a valuable approach to quantification of serum proteins. We also demonstrated the presence of differentially expressed serum proteins between non-metastatic and metastatic OSCCs that may be further validated as biomarkers for metastatic OSCC. However, in order to comprehensively quantify low abundant serum proteins, a more efficient approach is needed to deplete highly abundant proteins prior to quantitative serum proteome analysis of OSCC.

Potential Biomarkers in the Sera of Breast Cancer Patients from Bahawalpur, Pakistan

“…A variable expression pattern is also observed for some components of complement system including C3, C4 and C8. Differential expression of multiple proteins has been reported in the sera of colon cancer37 and metastatic oral cancer patients 38. In addition, a protein fraction containing apoA-1 and Ig kappa chain c-region is also pre-dominantly up-regulated in breast cancer and benign breast disease patients.…”

Analysis of the plasma proteome using iTRAQ and TMT‐based Isobaric labeling

“…Lukzac et al in their LC‐MALDI‐TOF/TOF based evaluation of the optimal methods for plasma sample pre‐treatment prior to quantitative analysis using iTRAQ labeling, concluded that SCX chromatography without affinity depletion was the best pre‐treatment method, reporting high reproducibility of the analysis of 1427 proteins . SDS‐PAGE has also been used as a pre‐fractionation method prior to labeling . As another alternative to change the range of protein abundance, solvent‐based precipitation methods have been used .…”

iTRAQ Analysis with Paul Ion Trap–Obstacle Solved

“…17% of the parent ion m / z , is a property of the Bruker's ion traps, we believe that methodology proposed by us can be easily adopted for other ion traps offered on the market. For the Thermo LTQ instruments, the method of iTRAQ analysis with peptide identification based on MS/MS scan and quantification of the tag in a subsequent MS 3 scan has been utilized using PQD. 7 …”

“…Several approaches were used to address this issue as different fragmentation methods have been developed, including Pulsed-Q dissociation (PQD), 3 high-energy C-trap dissociation (HCD), 4 electron transfer dissociation (ETD), 5 and MS 3 procedure 6 to facilitate the quantification of low mass reporter ions.…”