Quantitative LC-MS/MS Analysis of Proteins Involved in Metastasis of Breast Cancer

Abstract

The purpose of this study was to develop quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the analysis of proteins involved in metastasis of breast cancer for diagnosis and determining disease prognosis, as well as to further our understand of metastatic mechanisms. We have previously demonstrated that the protein type XIV collagen may be specifically expressed in metastatic tissues by two dimensional LC-MS/MS. In this study, we developed quantitative LC-MS/MS methods for type XIV collagen. Type XIV collagen was quantified by analyzing 2 peptides generated by digesting type XIV collagen using stable isotope-labeled peptides. The individual concentrations were equivalent between 2 different peptides of type XIV collagen by evaluation of imprecise transitions and using the best transition for the peptide concentration. The results indicated that type XIV collagen is highly expressed in metastatic tissues of patients with massive lymph node involvement compared to non-metastatic tissues. These findings were validated by quantitative real-time RT-PCR. Further studies on type XIV collagen are desired to verify its role as a prognostic factor and diagnosis marker for metastasis.

Fig 1. Average intra-assay concentration of peptide ITWDPPSSPVK and peptide ASAHAITGPPTELITSEVTAR of type XIV collagen.

Peptides from each subject (subjects 1–6: non metastasis, subjects 7–18: massive lymph node metastasis) were quantified (n = 3) by LC-MS/MS. The average intra-assay concentration and SD of peptide concentrations from individual proteins are calculated from all transitions (614/711, 614/826 and 614/1013) for peptide ITWDPPSSPVK of Protein 12 (A), 2 transitions (614/711 and 614/826) for peptide ITWDPPSSPVK (A'), and all transitions (708/624, 708/763 and 708/877) for peptide ASAHAITGPPTELITSEVTAR (B) of type XIV collagen. By excluding the low specificity transition 614/1013, the concentration of peptide ITWDPPSSPVK was changed (A) to (A'), which correlated with the concentrations of (B). Significant difference between non metastasis and massive lymph node metastasis in (A') and (B).

Fig 2. Average peak area of internal standards of peptide ITWDPPSSPVK and peptide ASAHAITGPPTELITSEVTAR.

The internal standards of peptide ITWDPPSSPVK and peptide ASAHAITGPPTELITSEVTAR were peptide I (¹³C₆, ¹⁵N) TWDPPSSPVK and peptide ASAHAI (¹³C₆, ¹⁵N) TGPPTELITSEVTAR, respectively. The peak areas of peptide I(¹³C₆, ¹⁵N)TWDPPSSPVK were 8% in subject 4 and 1% in subject 7, which were less than 10% of the values in other subjects (A). The peak area of peptide ASAHAI(¹³C₆, ¹⁵N)TGPPTELITSEVTAR was equivalent in all subjects.

Fig 3. MRM chromatograms for peptide ITWDPPSSPVK of subject 4and subject 7.

Transitions 614/711, 614/826 and 614/1013 were for endogenous peptide ITWDPPSSPVK, and transitions 617/711, 617/826, and 617/1013 were for stable isotope-labeled peptide, peptide I (¹³C₆, ¹⁵N) TWDPPSSPVK. (A) subject 4 (B) subject 7. Only the endogenous peptide area of transition 614/1013 in subjects 4 and 7 was as high as that in other subjects, while other transitions (614/711 or 614/826) showed no peaks.

Fig 4. Type XIV collagen concentrations calculated from the best transitions of peptide ITWDPPSSPVK and peptide ASAHAITGPPTELITSEVTAR.

After evaluation of imprecise transitions and using the best transition for the peptide concentration, concentrations of transition 614/711 were used as the concentrations of peptide ITWDPPSSPVK, and concentrations of transition 708/763 were used as the concentrations of peptide ASAHAITGPPTELITSEVTAR.

Fig 5. Correlation of type XIV collagen concentrations calculated from the best transitions of peptide ITWDPPSSPVK and peptide ASAHAITGPPTELITSEVTAR.

The x-axis represents protein concentration of type XIV collagen calculated from the best transition (708/763) of peptide ASAHAITGPPTELITSEVTAR as determined by LC-MS/MS. The y-axis represents protein concentration of type XIV collagen calculated from the best transition (614/711) of peptide ITWDPPSSPVK as determined by LC-MS/MS. In the scatter plot, each data point represents protein concentration.

Fig 6. Correlation of concentrations between LC-MS/MS and quantitative real-time RT-PCR for validation of type XIV collagen.

The x-axis represents m-RNA expression of type XIV collagen as determined by quantitative real-time RT-PCR. The y-axis represents protein concentration of type XIV collagen as determined by LC-MS/MS. In the scatter plot, each data point represents protein concentration and m-RNA expression. (A) The protein concentrations are average concentrations of all peptide transitions (614/711, 614/826, and 614/1013) of peptide ITWDPPSSPVK. (A') The protein concentrations are calculated from the best transition (614/711) for peptide ITWDPPSSPVK. (B) The protein concentrations are calculated from the best transition (708/763) for peptide ASAHAITGPPTELITSEVTAR.