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Deeper insight into chronic kidney disease-related atherosclerosis: comparative proteomic studies of blood plasma using 2DE and mass spectrometry.

Luczak M, Formanowicz D, Marczak Ł, Pawliczak E, Wanic-Kossowska M, Figlerowicz M, Stobiecki M - J Transl Med (2015)

Bottom Line: Differential accumulated proteins were confirmed by selected reaction monitoring analysis (SRM).For eleven of them differential expression were confirmed by selected reaction monitoring analysis.Undoubtedly, in advanced stages of CKD inflammation is highly pronounced.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704, Poznan, Poland. magdalu@ibch.poznan.pl.

ABSTRACT

Background: Atherosclerosis is a major cause of cardiac events and mortality in patients suffering from chronic kidney disease (CKD). Moreover, the risk of cardiovascular disease (CVD) development in patients with CKD increases as kidney function declines. Although the close connection between atherosclerosis and kidney dysfunction is undeniable, particular risk factors and specific mechanisms that promote CVD in patients with CKD remain unclear. To gain insight into better recognition of the mechanisms of accelerated atherosclerosis in patients with CKD, we performed a comparative proteomic analysis of blood plasma from patients in various stages of CKD and thus distinct progression of atherosclerosis (n = 90), patients with advanced CVD and normal renal function (n = 30) and healthy volunteers (n = 30).

Methods: Plasma samples were depleted using affinity chromatography and divided into three fractions: high-abundant, low-abundant and low-molecular weight proteins. The first two fractions were analyzed by two-dimensional gel electrophoresis and mass spectrometry, the last one has been subjected to direct MS/MS analysis. A proteomic profiles for high-abundant, low-abundant and low-molecular weight proteins fractions were obtained. Differential accumulated proteins were confirmed by selected reaction monitoring analysis (SRM). The Gene Ontology (GO) function and the interaction networks of differentially expressed proteins were then analyzed.

Results: Forty-nine proteins (13 high- and 36 low-molecular mass) showed differences in accumulation levels. For eleven of them differential expression were confirmed by selected reaction monitoring analysis. Bioinformatic analysis showed that identified differential proteins were related to three different processes: the blood coagulation cascade, the transport, binding and metabolism of lipoproteins and inflammatory processes.

Conclusions: Obtained data provide an additional line of evidence that different molecular mechanisms are involved in the development of CKD- and CVD-related atherosclerosis. The abundance of some anti-atherogenic factors revealed in patients with CKD suggests that these factors are not associated with the reduction of atherosclerosis progression in CKD that is typically observed in "classical" CVD. Moreover, obtained data also suggest that mechanism of CVD acceleration may be different in initial and advanced stages of CKD. Undoubtedly, in advanced stages of CKD inflammation is highly pronounced.

No MeSH data available.


Related in: MedlinePlus

An example of the SRM analysis of α-1 m. A. The extracted ion chromatograms of SRM transitions of the ETLLQDFR peptide of α-1 m in HV, CKD1-2, CKD3-4, CKD5 and CVD plasma. B. The MS/MS spectrum of the transition 511 - > 565.5 m/z (parent mass - > fragment mass).
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Fig4: An example of the SRM analysis of α-1 m. A. The extracted ion chromatograms of SRM transitions of the ETLLQDFR peptide of α-1 m in HV, CKD1-2, CKD3-4, CKD5 and CVD plasma. B. The MS/MS spectrum of the transition 511 - > 565.5 m/z (parent mass - > fragment mass).

Mentions: To confirmation of up- or down-regulation of proteins identified in 2DE/MS analysis, SRM experiments were performed. We first optimized the SRM method using nanoLC-ESI-IT MSn. This technique was then used to monitor the amounts of identified proteins in samples from each analyzed group. Using the SRM approach, we successfully validated the differential accumulation of ten proteins from the HAP and LAP fractions: α-1 m, APOA4, A2M, APOA1, CFB, ITIH4, TTR, FGG, HP and APOB and one protein identified in LMWP fraction (C4A13). For these proteins, we were able to obtain specific and “clean” transitions. SRM analyses confirmed the results obtained using 2DE for all proteins except one - KIN. Therefore, this protein was excluded from the list of differentially expressed proteins and from subsequent analysis. The 10 differentially expressed proteins that were successfully validated retained the same accumulation trends as observed in 2DE analysis (Table 2, Figure 2). For some proteins insignificant in the 2DE analysis, the SRM data showed statistical significance. For TTR, the SRM analysis showed more meaningful differences in the relative abundance compared with the 2DE analysis (i.e. for comparison between the CKD3-4 and HV groups, fold change for SRM was 1.89, instead of 1.23 for 2DE analysis). Differences in accumulation between HVs and CKD1-2 or CKD3-4 patients were more visible for CFB as well (respective fold changes of 1.78 and 1.87 instead of 1.43 and 1.48 in the 2DE analysis) (Table 2B). Although the magnitude of the relative ratios of the validated proteins differed between techniques used, the direction of change in expression for the validated protein spots was consistent between 2DE and SRM analyses. It should be noted that differences in the accumulation of particular proteins were always more pronounced in SRM analysis compared with 2DE analysis. Examples of the SRM analysis for α-1 m and APOA4 peptides are shown in Figures 4 and 5.Figure 4


Deeper insight into chronic kidney disease-related atherosclerosis: comparative proteomic studies of blood plasma using 2DE and mass spectrometry.

Luczak M, Formanowicz D, Marczak Ł, Pawliczak E, Wanic-Kossowska M, Figlerowicz M, Stobiecki M - J Transl Med (2015)

An example of the SRM analysis of α-1 m. A. The extracted ion chromatograms of SRM transitions of the ETLLQDFR peptide of α-1 m in HV, CKD1-2, CKD3-4, CKD5 and CVD plasma. B. The MS/MS spectrum of the transition 511 - > 565.5 m/z (parent mass - > fragment mass).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4316657&req=5

Fig4: An example of the SRM analysis of α-1 m. A. The extracted ion chromatograms of SRM transitions of the ETLLQDFR peptide of α-1 m in HV, CKD1-2, CKD3-4, CKD5 and CVD plasma. B. The MS/MS spectrum of the transition 511 - > 565.5 m/z (parent mass - > fragment mass).
Mentions: To confirmation of up- or down-regulation of proteins identified in 2DE/MS analysis, SRM experiments were performed. We first optimized the SRM method using nanoLC-ESI-IT MSn. This technique was then used to monitor the amounts of identified proteins in samples from each analyzed group. Using the SRM approach, we successfully validated the differential accumulation of ten proteins from the HAP and LAP fractions: α-1 m, APOA4, A2M, APOA1, CFB, ITIH4, TTR, FGG, HP and APOB and one protein identified in LMWP fraction (C4A13). For these proteins, we were able to obtain specific and “clean” transitions. SRM analyses confirmed the results obtained using 2DE for all proteins except one - KIN. Therefore, this protein was excluded from the list of differentially expressed proteins and from subsequent analysis. The 10 differentially expressed proteins that were successfully validated retained the same accumulation trends as observed in 2DE analysis (Table 2, Figure 2). For some proteins insignificant in the 2DE analysis, the SRM data showed statistical significance. For TTR, the SRM analysis showed more meaningful differences in the relative abundance compared with the 2DE analysis (i.e. for comparison between the CKD3-4 and HV groups, fold change for SRM was 1.89, instead of 1.23 for 2DE analysis). Differences in accumulation between HVs and CKD1-2 or CKD3-4 patients were more visible for CFB as well (respective fold changes of 1.78 and 1.87 instead of 1.43 and 1.48 in the 2DE analysis) (Table 2B). Although the magnitude of the relative ratios of the validated proteins differed between techniques used, the direction of change in expression for the validated protein spots was consistent between 2DE and SRM analyses. It should be noted that differences in the accumulation of particular proteins were always more pronounced in SRM analysis compared with 2DE analysis. Examples of the SRM analysis for α-1 m and APOA4 peptides are shown in Figures 4 and 5.Figure 4

Bottom Line: Differential accumulated proteins were confirmed by selected reaction monitoring analysis (SRM).For eleven of them differential expression were confirmed by selected reaction monitoring analysis.Undoubtedly, in advanced stages of CKD inflammation is highly pronounced.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704, Poznan, Poland. magdalu@ibch.poznan.pl.

ABSTRACT

Background: Atherosclerosis is a major cause of cardiac events and mortality in patients suffering from chronic kidney disease (CKD). Moreover, the risk of cardiovascular disease (CVD) development in patients with CKD increases as kidney function declines. Although the close connection between atherosclerosis and kidney dysfunction is undeniable, particular risk factors and specific mechanisms that promote CVD in patients with CKD remain unclear. To gain insight into better recognition of the mechanisms of accelerated atherosclerosis in patients with CKD, we performed a comparative proteomic analysis of blood plasma from patients in various stages of CKD and thus distinct progression of atherosclerosis (n = 90), patients with advanced CVD and normal renal function (n = 30) and healthy volunteers (n = 30).

Methods: Plasma samples were depleted using affinity chromatography and divided into three fractions: high-abundant, low-abundant and low-molecular weight proteins. The first two fractions were analyzed by two-dimensional gel electrophoresis and mass spectrometry, the last one has been subjected to direct MS/MS analysis. A proteomic profiles for high-abundant, low-abundant and low-molecular weight proteins fractions were obtained. Differential accumulated proteins were confirmed by selected reaction monitoring analysis (SRM). The Gene Ontology (GO) function and the interaction networks of differentially expressed proteins were then analyzed.

Results: Forty-nine proteins (13 high- and 36 low-molecular mass) showed differences in accumulation levels. For eleven of them differential expression were confirmed by selected reaction monitoring analysis. Bioinformatic analysis showed that identified differential proteins were related to three different processes: the blood coagulation cascade, the transport, binding and metabolism of lipoproteins and inflammatory processes.

Conclusions: Obtained data provide an additional line of evidence that different molecular mechanisms are involved in the development of CKD- and CVD-related atherosclerosis. The abundance of some anti-atherogenic factors revealed in patients with CKD suggests that these factors are not associated with the reduction of atherosclerosis progression in CKD that is typically observed in "classical" CVD. Moreover, obtained data also suggest that mechanism of CVD acceleration may be different in initial and advanced stages of CKD. Undoubtedly, in advanced stages of CKD inflammation is highly pronounced.

No MeSH data available.


Related in: MedlinePlus