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Glycoproteomic Analysis of Human Lung Adenocarcinomas Using Glycoarrays and Tandem Mass Spectrometry: Differential Expression and Glycosylation Patterns of Vimentin and Fetuin A Isoforms

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Abstract

Human lung cancer is a major cause of cancer mortality worldwide. Advances in pathophysiologic understanding and novel biomarkers for diagnosis and treatment are significant tasks. We have undertaken a comprehensive glycoproteomic analysis of human lung adenocarcinoma tissues. Glycoproteins from paired lung adenocarcinoma and normal tissues were enriched by the lectins Con A, WGA, and AIL. 2-D PAGE revealed 30 differentially expressed protein spots, and 15 proteins were identified by MS/MS, including 8 up- (A1AT, ALDOA, ANXA1, CALR, ENOA, PDIA1, PSB1 and SODM) and 7 down-regulated (ANXA3, CAH2, FETUA, HBB, PRDX2, RAGE and VIME) proteins in lung cancer. By reverse-transcription PCR, nine proteins showed positive correlation between mRNA and glycoprotein expression. Vimentin and fetuin A (α2-HS-glycoprotein) were selected for further investigation. While for vimentin there was little correlation between total protein and mRNA abundance, expression of WGA-captured glycosylated vimentin protein was frequently decreased in cancer. Glycoarray analysis suggested that vimentins from normal and cancerous lung tissue differ in their contents of sialic acid and terminal GlcNAc. For fetuin A, both total protein and mRNA abundance showed concordant decrease in cancer. WGA- and AIL-binding glycosylated fetuin A was also consistently decreased in cancer. Glycoarray analysis suggested that high mannose glycan structures on fetuin A were only detectable in cancer but not normal tissue. The intriguing expression patterns of different isoforms of glycosylated vimentin and fetuin A in lung cancer illustrate the complexities and benefits of in-depth glycoproteomic analysis. In particular, the discovery of differentially glycosylated protein isoforms in lung adenocarcinoma may represent avenues towards new functional biomarkers for diagnosis, treatment guidance, and response monitoring.

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Abbreviations

Con A:

Concanavalin A

WGA:

Wheat germ agglutinin

AIL:

Amylase inhibitor-like protein (jacalin)

CALR:

Calreticulin

VIME:

Vimentin

FETUA:

Fetuin A (α2-HS-glycoprotein)

A1AT:

α1-Antitrypsin

PDIA1:

Protein disulfide isomerase A1

PRDX2:

Peroxiredoxin-2

ANXA1:

Annexin A1

ANXA3:

Annexin A3

RAGE:

Receptor for advanced glycosylation end products

SODM:

Mitochondrial superoxide dismutase

CAH2:

Carbonic anhydrase 2

ENOA:

α-Enolase

HBB:

Hemoglobin subunit β

PSB1:

Proteasome subunit β type 1

ALDOA:

Fructose-bisphosphate aldolase A

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Acknowledgments

We acknowledge financial support from the NIAID/NIH. We thank Drs. Bin Ye, Brian Liu, and Sam Mok of Brigham and Women’s Hospital for use of proteomic equipment. We thank the Taplin Biological Mass Spectrometry Center of Harvard Medical School for expert advice.

Author information

Authors and Affiliations

  1. Channing Laboratory, Department of Medicine, Brigham and Women’s Hospital, Boston, MA, 02115, USA

    Jung-Hyun Rho & Julia Y. Wang

  2. Department of Pathology, Massachusetts General Hospital, 55 Fruit Street, Boston, MA, 02114, USA

    Michael H. A. Roehrl

  3. Harvard Medical School, Boston, MA, 02115, USA

    Jung-Hyun Rho, Michael H. A. Roehrl & Julia Y. Wang

Authors
  1. Jung-Hyun Rho
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  2. Michael H. A. Roehrl
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  3. Julia Y. Wang
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Corresponding author

Correspondence to Michael H. A. Roehrl.

Electronic supplementary material

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Supplementary Table 1

Detailed information on the MS/MS sequencing of the identified protein spots. (DOCX 68 kb)

Supplementary Fig. 1

2-D PAGE profiles of lectin-captured proteins from patient 4. (PDF 382 kb)

Supplementary Fig. 2

Magnified paired views of the 16 protein spots (arrowheads) from representative patient samples sequenced by MS/MS (N, normal lung; C, cancer). Spots 13 and 22 yielded the same protein identity (RAGE). (PDF 430 kb)

Supplementary Fig. 3

Comparison of mRNA expression levels between paired normal and lung adenocarcinoma tissues for 11 selected glycoproteins. RT-PCR analysis was performed on matched normal/tumor pairs from 7 patients (N/D, not detected). Data for the remaining 3 glycoproteins are shown in Fig. 2. RPL13A (ribosomal protein L13A) was used as a control transcript. The tumor to normal (T:N) ratio of each sample was normalized relative to the corresponding control transcript ratio. Amplification of ANXA3 (Fig. 1, spot 9) mRNA was not successful despite attempts with two sets of PCR primers. The average mRNA ratios (T:N) are shown in Table 1. (PDF 263 kb)

Supplementary Fig. 4

Confirmation of glycosylation and comparison of total protein abundance of vimentin and fetuin A. Presence of N-glycosylation of vimentin (A) and fetuin A (B) was demonstrated by PNGase F digestion. Unfractionated total soluble proteins were digested with PNGase F ((-), protein samples before PNGase F treatment; P, PNGase F-treated samples). Vimentin showed several protein bands. The most dominant band was selected for mobility shift calculation. Fetuin A showed two bands, both of which were shifted after deglycosylation to a similar extent. The calculated molecular weights (in kDa) of the vimentin bands are: A, 50.6; A’, 49.2; B, 50.6; B’, 49.7; C, 50.7; C’, 49.0; D, 50.6; D’, 49.5. The calculated molecular weights (in kDa) of the fetuin A bands are: E, 48.5; E’, 46.0; e, 44.1; e’, 42.3; F, 48.2; F’, 46.0; G, 48.6; G’, 46.3; H, 48.6; H’, 46.3. (C) Western blot comparison of total vimentin and fetuin A proteins. Unfractionated lung tissue protein extracts were used. The total intensities of all isoforms were used for comparison. Normalized expression ratios (T:N) are shown below the protein bands using (-actin the as an internal control. Ratios of >1, 1, or <1 describe increased, unchanged, or decreased expression in cancer, respectively. (PDF 300 kb)

Supplementary Fig. 5

Representative lectin glycoarray images. The fingerprint patterns of glycosylated vimentin (top) or fetuin A (bottom) isolated from cancer tissue protein extracts are shown. (PDF 236 kb)

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Rho, JH., Roehrl, M.H.A. & Wang, J.Y. Glycoproteomic Analysis of Human Lung Adenocarcinomas Using Glycoarrays and Tandem Mass Spectrometry: Differential Expression and Glycosylation Patterns of Vimentin and Fetuin A Isoforms. Protein J 28, 148–160 (2009). https://doi.org/10.1007/s10930-009-9177-0

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