Table 1.
The Applications of MALDI-MS and SELDI-MS for Preeclampsia and Preterm Birth.
| Technique | Sample | Cohort | Sample Preparation | Results | Biological Implications | Ref. |
|---|---|---|---|---|---|---|
| MALDI-MS(ClinProt) | Serum | Early-onset sPE: 11 CRL: 13 |
Sample extraction and enrichment was performed on HIC8 reverse phase coated magnetic beads. 5 μL of serum was incubated with 10 μL MB-HIC8 binding buffer and 5 μL of MB-HIC8 bead slurry for 1 min. After washing twice with 100 μL of wash buffer, proteins were eluted with 10 μL of elution buffer. After MB-HIC8 extraction, protein solutions, 0.5 μL of protein solution was spotted directly onto a stainless steel target plate before matrix solution was added. | The best differentiating signals between the two sample groups were found at m/z 13,715, 13,834, and 13,891. The normalized intensities of these ion signals were on average lower in the PE group than in the control group. The ion signals were believed to belong to the protein transthyretin and its modified forms. Results were consistent with that obtained with SDS-PAGE analysis. | The reduction of transthyretin concentrations is expected during pregnancy due to plasma volume expansion. Transthyretin is synthesized by the liver and also secreted by placental trophoblasts where it binds extracellular T4, which in turn result in an increased internalization of the transthyretin-T4 complex. It has been suggested that transthyretin plays an important role in the transfer of maternal thyroid hormone to the fetal circulation, which could have important implications for fetal development. | [75] |
| SELDI-MS (WCX2 array) | Cerebrospinal fluid (CSF) | sPE: 7 mPE: 8 CRL: 8 |
Dry on-chip protocol: 5-μL of undiluted pooled CSF was dried onto individual spots. | A cluster of 4 peaks was observed in the 15–16.3 kDa region only from the CSF of the patients. These peaks were assigned to α- and β-chains of hemoglobin, and their glycosylated formed. The presence of hemoglobin in CSF as biomarkers was validated with ELISA and spectrophotometry. | The reason for the observations was not clear. The authors suggested that the increase in CSF hemoglobin might result from increased and selective trafficking of intact erythrocytes across the blood-brain barrier where they subsequently lyse releasing their hemoglobin content. | [65] |
| SELDI-MS (Q10 array) | Amniotic fluid (AF) | PE: 18 CHTN: 7 CRL: 16 |
Before sample loading, each ProteinChip spot was incubated twice with 5 µL binding buffer in a humidity chamber at room temperature for 5 min. After equilibration, 5 µL of sample, diluted 1:3 in binding buffer, was added to each spot and incubated in a humidity chamber with shaking for 40 min. Each spot was washed with 5 µL binding buffer for 2 min, followed by washing with 5 µL of triple distilled water, and air-dried. | 2 peaks located at 17,399.1 and 28,023.3 Da were significantly different. The former peak distinguished women with PE from control, and the latter peak distinguished women with PE and CHTN from controls. The peaks were assigned to hypothetical protein SBBI42 and proapolipoprotein A-I. The results were cross-validated with Western blot. | It was suggested that the increase in levels of proapolipoprotein A-I in the AF of women with PE may represent a compensatory mechanism to maintain levels of apolipoprotein A-I and thereby pulmonary surfactant and lung compliance and development. | [70] |
| SELDI-MS (H50 array) | Amniotic fluid (AF) | PE: 10 CRL:10 |
AF was obtained by transabdominal amniocentesis. Protein chip arrays were placed in a bioprocessor and pre-treated with 50% methanol for 5 min. 2 μL of AF and 3 μL of protein buffer were placed on individual sample spots and incubated at room temperature for 30 min. Each sample spot was equilibrated by adding 200 μL of binding buffer. After shaking for 5 min at room temperature, the buffer was removed, then, 5 μL of sample mixture and 195 μL of binding buffer were added to each spot and incubated with vigorous shaking for 30 min. The arrays were washed with 3 times of 200 μL binding buffer, followed by 2 times of 200 μL distilled water. The protein chip arrays were then air-dried. | 5 protein peaks located at m/z 4679, 9080, 14,045, 14,345 and 28,087 were significantly differentially expressed between case and control groups. The peak located at m/z 14,345 was characterized as fragmented albumin, whereas the peak located at m/z 28,087 was identified as apolipoprotein A-I. The increased expression of apolipoprotein A-I was confirmed with ELISA. | The reason why albumin fragment may be overexpressed in these women is not immediately apparent. One possibility is that the increased proteolytic activity against albumin is an early phenomenon that precedes the clinical manifestations of the disease. Apolipoprotein A-I is expressed in the placenta and acts as a receptor for cholesterol, which is then transferred to the fetus. The origin of apolipoprotein A-I within AF remains unclear. Even less well understood is why apolipoprotein A-I is overexpressed in second trimester AF of women destined to develop pre-eclampsia. Whether the increase in apolipoprotein A-I in the AF precedes the increase seen in maternal plasma and urine in women with pre-eclampsia is not known. | [71] |
| SELDI-MS (Q10 array) | Urine | sPE: 11 mPE: 7 CRL: 8 |
30 μL aliquots of individual urine samples were mixed with 10 μL of sample buffer. Following 30 min incubation at room temperature, 160 μL of binding buffer was added to each sample. After equilibration of the ProteinChip Array, 150 μL of diluted sample mixture was loaded onto each spot and incubated with vigorous shaking for 1 h. Each spot was washed and air-dried. | 4 discriminatory protein peaks were identified at m/z 4155, 6044, 6663 and 7971. All of these proteins had a lower concentration in urine. The identities of the protein biomarkers were not assigned, but their discriminatory power was tested with ROC. | [72] | |
| SELDI-MS (H4 and H50 array) | Urine | sPE: 38 CRL: 21 |
ProteinChip arrays were incubated for 1 h with the samples (6 μL/spot) diluted to 0.25 mg/mL total protein. Following incubation, unbound proteins were removed by washing each spot with the respective buffer and dried. | At the end of exploratory phase, urine proteomic profiles from the patients with sPE exhibited 13 peaks qualitatively different from that of the controls. These peaks were assigned to non-random cleavage products of serpin peptidase inhibitor-1 (SERPINA1) and albumin protein. Urine proteomic profile score was tested against a cross-sectional cohort (n = 206). Performance was evaluated by ROC (AUC = 0.92). The over-expression of SERPINA1 in urine was also verified with western blot. | Other studies have shown that minor increases in levels of serum SERPINA1 are associated with the development of arterial hypertension and an increased risk of cardiovascular disease. The authors suggested that by inhibiting the activity of the kallikrein-kinin system, an up-regulation of plasma SERPINA1 favors the renin-angiotensin system, leading to systemic vasoconstriction and hypertension. Urinary albumin excretion is a hallmark of PE. | [74] |
| SELDI-MS (NP20, H4 and IMAC array) | Amniotic fluid (AF) | Preterm +IAI: 11 Preterm only: 11 CRL: 11 |
0.5 to 3.0 µg of unfractionated protein from AF was deposited on 3 different ProteinChip arrays (normal-phase SiO2, a reverse-phase hydrophobic, and immobilized nickel surfaces). The Chips were incubated for 1 hour with the sample followed by a 5-µL water wash, and subsequently dried. | A set of peaks located at 10- to 12-kDa was differentially expressed. The peaks were observed on all 11 patients with subclinical IAI, in 2 of 11 with preterm delivery without IAI, and in 0 of 11 with preterm labor and term delivery without infection. The signatures were identified polypeptides derived from calgranulin B and a unique fragment of insulin-like growth factor binding protein 1 (IGFBP-1). Results were validated by western blot. | The calgranulins are members of the S-100 calcium-binding protein family, expressed by macrophages and by epithelial cells in acutely inflamed tissues. The second candidate from this cluster, a specific proteolytic fragment of IGFBP-1, indicates a potential protease-related mechanism in response to infection. Intact IGFBP-1 is the major IGFBP found in AF and is synthesized by both fetal membranes and maternal decidua. | [66] |
| SELDI-MS (H4 array) | Amniotic fluid (AF) | Preterm (+WBC; +AFC): 21 Preterm (+WBC; −AFC): 7 Preterm (−WBC; +AFC): 8 Preterm (−WBC; −AFC): 24 CRL: 17 |
2 μL of AF diluted 10-fold in PBS. After 1-h incubation in a humidified box, the sample was aspirated and the spots washed individually with 25% aqueous acetonitrile solution, air-dried. | Candidate makers were tested on a separate set of 24 samples by blinding independent examiners to the outcomes. 3 additional samples were used to assess the possibility of storage artefacts and to calculate intra- and inter-rater agreement among the 3 investigators. 4 proteins, neutrophil defensins-1 and -2, and calgranulins A and C were found distinctive and were validated with western blot. | Neutrophil defensins (α-defensins) belong to a family of cationic antimicrobial peptides. These key components of the host-defense mechanism exert their bactericidal activity by punching pores into bacterial membranes. | [67] |
| SELDI-MS (RS100) | Amniotic fluid (AF) | Preterm +IAI: 86 Preterm only: 86 CRL: 86 |
5 μL of anti-IGFBP-1 antibody or control IgG solutions was loaded onto the spot of pre-activated ProteinChip arrays and covalently coupled for 2 h at room temperature in a humidity chamber. Remaining reactive groups were blocked for 1 h with 2 mg/mL BSA in 50 mM Tris-HCl. The spots were washed 3 times with 10 μL of PBS. Then, 5 μL AF samples were loaded on the antibody-coated arrays and incubated for 1 h in the humidity chamber. Arrays were washed three times with PBS and rinsed once with water before air-drying. | The ProteinChip array-based immunoassay using SELDI showed that IGFBP-1 was largely in a full-length form in the AF of the patients with preterm labor without IAI, but significantly degraded in the AF pool of the patients who delivered preterm with IAI. This indicated a preferential production of IGFBP-1 fragments in the amniotic fluid of patients with IAI. | Consistent with the previous study that the proteolytic degradation of IGFBP-1 by matrix metalloproteinases (MMPs) and different MMPs generated fragments of IGFBP-1 of different masses. | [68] |
| SELDI-MS (CM10 and H50) | Amniotic fluid (AF) | Preterm +IAI: 60 CRL: 59 |
AF from each patient was diluted in sterile PBS at a 1:10 dilution and was added onto the ProteinChip. | 39 peaks were distinguishing patients with preterm labor with IAI from those with preterm lab our but subsequently delivered at term. The study did not seek to identify these mass spectrometric features. | [69] |
Q10: strong anion exchange; CM10/WCX: weak cation exchange; H: hydrophobic (reverse-phase); CRL: controls; PE: preeclampsia; sPE: severe preeclampsia; mPE: mild preeclampsia; CHTN: chronic hypertension; +IAI: intra-amniotic infection; +AFC: positive amniotic fluid culture results; +WBC: white blood cell count > 100 cells/mm3; PBS: phosphate buffered saline; BSA: bovine serum albumin.