As an example, Fig. 3 illustrates a comparison of multiple biomarkers in serum fraction 1 (pH 9.0 eluate) of SARS patients vs all the other controls in a gel view format in the m/z range of 2000–15,000.
Highlighted in three boxes (A, B, and C) with arrows are biomarkers at m/z of 11,695, 9159, and 7784, respectively with biomarker at 11,695 being significantly increased in the SARS patients vs the controls and biomarkers 9159 and 7784 being significantly decreased.
Nine biomarker were found within m/z range of 4900–15,000 with highly significant increase in their normalized peak intensities in the SARS patients vs those of the controls and three biomarkers with highly significant decrease (Table 1).
Separating the controls into individual groups, a highly significant increase of biomarker at 11,695 is also observed in SARS patients vs each control group (Fig. 4).
As pneumonia is a frequent life threatening clinical manifestation in the SARS patients, we further monitor the biomarker—11,695 level in a SARS patient longitudinally (Fig. 5) and correlate the biomarker level with the extent of pneumonia by chest X-ray opacity score (Fig. 2).
Figure 5 illustrates, in this patient, an elevation of the biomarker—11,695 level preceding the development of pneumonia at the onset of disease (as indicated by an increasing opacity score) followed by a rapid drop of the biomarker level down to the background on recovery from the illness after stringent antiviral and steroid treatments (seeNote 1 for details of clinical presentation of the patients).
On the other hand, Fig. 6 shows low biomarker—11,695 level during the whole monitoring period in another SARS patient who has low X-ray scores and whose clinical course is relatively mild (see Note 2).
By means of peptide mapping and tandem MS/MS mass spectrometry, this biomarker is identified to be Serum Amyloid A protein (SAA), which is an acute phase reactant frequently and rapidly induced in abundance at pneumonia (see ref. 15 for the protein ID results and ref. 17–20 for the techniques adopted).
This serial study demonstrates the potential of the biomarkers discovered by protein chip array profiling in monitoring the disease manifestation in SARS patients.
Fig. 3. Gel view of partial protein chip array profiling results in SARS patients’ sera vs control infection groups’ sera. Part of the profiling results of fraction 1 from the ion exchange serum eluate in SARS patients vs the control patients in m/z range from 2000 to 15,000 was illustrated. HBV, hepatitis B virus; TB, M. tuberculosis; RSV, respiratory syncytial virus. The three arrows showed three clusters of biomarkers, A–C at m/z of 11695, 9159, and 7784, respectively, with cluster A being significantly increased in SARS patients and the last two clusters (B and C) significantly decreased.
Table 1  Serum Biomarkers Significantly Increased or Decreased in SARS Patients vs Seven Control Groups of Patients With Other Infections and Healthy Individuals
Biomarker number Mass over charge (m/z) Differential expression p-valuea
1 4922 Increased 1.1 × 10−4
2 5104 Increased 7.1 × 10−8
3 5215 Increased 6.4 × 10−4
4 5833 Increased 3.1 × 10−9
5 7784 Decreased 4.9 × 10−8
6 8416 Decreased 1.6 × 10−7
7 9159 Decreased 1 × 10−10
8 10,867 Increased 3.6 × 10−7
9 11,508 Increased 1.3 × 10−5
10 11,695 Increased 5.4 × 10−6
11 11,871 Increased 8.4 × 10−4
12 14,715 Increased 3.9 × 10−9
ap-values were obtained in Mann-Whitney U-test by comparing the normalized peaki intensities of the biomarkers in 44 sera from 24 SARS patients vs those in 72 sera from 51 control patients with various viral or bacterial infections (see Materials for individual control group) plus 10 sera from 10 apparently healthy individuals.
Fig. 4. Comparison of normalized peak intensities of the biomarker at m/z of 11,695 in sera from SARS patients and control groups. The normalized peak intensities of this marker in SARS patients’ sera were compared with those of the other seven control infection groups and the normal individuals by Mann-Whitney U-test. A p-value of 0.05 or less demonstrated a statistically significant difference between the two groups. Adeno, adenovirus; Bact, bacterial culture positive; HBV, hepatitis B virus; Influ A, influenza A virus; Influ B, influenza B virus; Normal, apparently normal subjects; RSV, respiratory syncytial virus; TB, M. tuberculosis.
Fig. 5. Correlation of the level of serum biomarker—11,695 with the extent of pneumonia in a SARS patient under clinical follow-up. Longitudinal follow- up of the clinical profile of this patient was illustrated in the top panel. Monitoring of the biomarker—11,695 level and chest X-ray opacity score (an indicator of the extent o pneumonia) was shown in the lower left panel. The lower right panel illustrates the biomarker—11,695 in a gel view measured at different time points. CoV IgG <25 and 100, Serum SARS-Coronavirus IgG antibody titers of <1/25 and 1/100; CXR, Lung consolidation as shown by chest X-ray imaging; FU outpatient, During follow-up as outpatient; ICU, Admitted to Intensive care unit; i.v. Ig, Intravenous injection of immune globulin; MP, Daily treatment by 500 mg × 2 of methylprednisolone; NP Swab CoV −ve, RT-PCR negative for SARS Coronavirus in nasopharyngeal swab; Rectal Swab CoV +ve, RT-PCR positive for SARS Coronavirus in rectal swab; Ribavirin, Daily treatment by 400 mg Ribavirin; Stool CoV +ve, RT-PCR positive for SARS Coronavirus in the stool; Throat Swab CoV +ve, RT PCR positive for SARS Coronavirus in throat swab.
Fig. 6. Correlation of the level of serum biomarker—11,695 with the extent of pneumonia in the second SARS patient under clinical follow-up. Longitudinal follow-up of the clinical profile of this patient was illustrated in the top panel. Monitoring of the biomarker—11,695 level and chest X-ray opacity score was shown in the lower panel. Annotations were similar to those as tabulated in Fig. 5. Antibiotic, Treatment with conventional antibiotics; CoV IgG <10 and 160, Serum SARS-Coronavirus IgG antibody titers of <1/10 and 1/160; NP Swab CoV −ve, RT-PCR negative for SARS Coronavirus in nasopharyngeal swab; Rectal Swab CoV −ve, RT-PCR negative for SARS Coronavirus in rectal swab; Throat Swab CoV −ve, RT PCR negative for SARS Coronavirus in throat swab.