The use of proteomics in identifying differentially expressed serum proteins in humans with type 2 diabetes

Abstract

Background: The aim of the study was to optimize protocols for finding and identifying serum proteins that are differentially expressed in persons with normal glucose tolerance (NGT) compared to individuals with type 2 diabetes mellitus (T2DM). Serum from persons with NGT and persons with T2DM was profiled using ProteinChip arrays and time-of-flight mass spectra were generated by surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS).

Results: Mass spectra from NGT- and T2DM-groups were compared. Fifteen proteins ranging from 5 to 79 kDa were differentially expressed (p < 0.05). Five of these proteins showed decreased and ten showed increased serum levels in individuals with T2DM. To be able to identify the proteins, the complexity of the sample was reduced by fractionation approaches. Subsequently, the purified fractions containing biomarkers were separated by one-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in two identical lanes. Protein bands of the first lane were excised and subjected to passive elution to recapture the biomarkers on ProteinChip arrays. The corresponding bands of the second lane were subjected to peptide-mass fingerprinting (PMF). Using this approach four of the differentially expressed proteins were identified as apolipoprotein C3 (9.4 kDa), transthyretin (13.9 kDa), albumin (66 kDa) and transferrin (79 kDa). Whereas apolipoprotein C3 and transthyretin were up-regulated, albumin and transferrin were down-regulated in T2DM.

Conclusion: Protocols for protein profiling by SELDI-TOF MS and protein identification by fractionation, SDS-PAGE and PMF were optimized for serum from humans with T2DM. With these protocols differentially expressed proteins were discovered and identified when serum from NGT- and T2DM-individuals was analyzed.

Figure 1

Mass spectra showing the differentially expressed proteins. SELDI-TOF mass spectra showing the six differentially expressed serum proteins captured by cationic exchanger array. The green spectra are from individuals with normal glucose tolerance and the red spectra are from individuals with type 2 diabetes mellitus.

Figure 2

Mass spectra of serum fractions showing the 9443 Da biomarker. SELDI-TOF mass spectra of serum after fractionation. Upper trace shows spectrum of the anionic fraction 5. Middle trace shows spectrum of the reverse phase beads fraction with 20 % acetonitrile. Lower trace shows the reverse phase beads fraction with 30 % acetonitrile. The abundant 66 kDa protein (albumin) was reduced in reverse phase fraction with 20 % acetonitrile (hatched area). The arrow indicates the 9443 Da biomarker.

Figure 3

SDS-PAGE of serum fractions. Proteins of serum fractions containing biomarkers were separated with one-dimensional gel electrophoresis. The two reverse phase beads (RPB) fractions containing 20 and 30 % acetonitrile are shown after staining with colloidal coomassie. Bands in the relevant mass range were excised and eluted. The arrows indicate the bands containing the 9443 biomarker.

Figure 4

Mass spectrum verifying the 9443 Da biomarker. Eluates were re-applied on the arrays to confirm that the eluted protein was the discovered biomarker at 9443 Da.