Quantitative Approach for Protein Analysis in Small Cell Ensembles by an Integrated Microfluidic Chip with MALDI Mass Spectrometry

Abstract

Increasing evidence has demonstrated that cells are individually heterogeneous. Advancing the technologies for single-cell analysis will improve our ability to characterize cells, study cell biology, design and screen drugs, and aid cancer diagnosis and treatment. Most current single-cell protein analysis approaches are based on fluorescent antibody-binding technology. However, this technology is limited by high background and cross-talk of multiple tags introduced by fluorescent labels. Stable isotope labels used in mass cytometry can overcome the spectral overlap of fluorophores. Nevertheless, the specificity of each antibody and heavy-metal-tagged antibody combination must be carefully validated to ensure detection of the intended target. Thus, novel single-cell protein analysis methods without using labels are urgently needed. Moreover, the labeling approach targets already known motifs, hampering the discovery of new biomarkers relevant to single-cell population variation. Here, we report a combined microfluidic and matrix-assisted laser desorption and ionization (MALDI) mass spectrometric approach for the analysis of protein biomarkers suitable for small cell ensembles. All necessary steps for cell analysis including cell lysis, protein capture, and digestion as well as MALDI matrix deposition are integrated on a microfluidic chip prior to the downstream MALDI-time-of-flight (TOF) detection. For proof of principle, this combined method is used to assess the amount of Bcl-2, an apoptosis regulator, in metastatic breast cancer cells (MCF-7) by using an isotope-labeled peptide as an internal standard. The proposed approach will eventually provide a new means for proteome studies in small cell ensembles with the potential for single-cell analysis and improve our ability in disease diagnosis, drug discovery, and personalized therapy.

Intracellular Amyloid-β Detection from Human Brain Sections Using a Microfluidic Immunoassay in Tandem with MALDI-MS

“…The absence of the peaks in the control experiments without an antibody and using a nonbinding antibody (IgG A11 specific for Aβ-O, which does not bind Aβ-M) confirmed minimal nonspecific binding. A peak with m/z ∼ 3880 was observed in all cases, which, as previously reported, is associated with the BSA blocking step. − The spectra from the Aβ 1–40 -M and Aβ 1–42 -M immunoassay in the MIMAS platform are shown in Figure . As in the milli-well immunoassay, peaks corresponding to [Aβ + H] + and [Aβ + 2H] 2+ were observed for Aβ 1–40 -M and Aβ 1–42 -M solutions exposed to IgG 6E10, and absent in the controls, confirming the lack of nonspecific binding (Figure S8).…”

“…In addition to the peak with m/z ∼ 3880 associated with the BSA blocking step often observed in the MIMAS assays, − peaks at m/z ∼ 66,000, ∼33,000, ∼22,000, and ∼ 16,500 were also observed (see the representative spectrum in Figure S10). The m/z 3880 peak related to the blocking step has been observed through the development of this microfluidic approach, − typically performed using a saturated α-HCCA solution as the matrix (as we typically assessed peptides <5 kDa). It is known that matrix selection and optimization are critical for assay development and can significantly impact analyte ionization. − Here, we used the sinapinic acid matrix for Aβ-O assays (recommended for >10 kDa molecules), which may explain the appearance of peaks that can be related to single and multiple-charged species as well as to the intact BSA molecule (MW 66 kDa) used in the blocking step.…”

“…The immunocapture of Aβ was performed in milli-wells and MIMAS wells following an adapted, previously reported protocol for on-chip protein immunocapture and MS analysis. − For the MIMAS platform, solution loading consists of valve opening by applying vacuum to the control layer inlets, allowing the solutions to fill up the wells by capillary action. The solutions were removed from the wells by opening the valves and applying a vacuum to the control layer outlets.…”

“…Here, with the aim of studying intracellular Aβ species from human brain tissues, we report the development of a microfluidic assay for in situ mass spectrometry of immunocaptured Aβ species from archived human brain tissues. We previously reported a microfluidic platform in tandem with MALDI mass spectrometry (MIMAS) for high-sensitivity detection and quantification of Bcl-2 protein in cultured MCF-7 breast cancer cells. − The reported MIMAS platform allows for cell lysis on-chip using freeze–thaw cycles, immunocapture of the target protein on-chip, trypsin digestion on-chip, and by using an isotope-labeled peptide of one of the Bcl-2 tryptic fragments, quantification of the Bcl-2 protein from as low as 10 cultured cells. , Preliminary work showed the capability of dissecting and collecting tissue cells directly on microfluidic wells, including the preparation and immunocapture of Aβ monomeric and oligomeric species…”

What the Microscale Systems “See” In Biological Assemblies: Cells and Viruses?

“…Therefore, it is necessary to study the cytotoxicity mechanism and cellular distribution of FePt NPs. The microchip–MS system is considered an efficient platform for the cell assay. , A droplet-splitting microchip integrated with time-resolved inductively coupled plasma MS (ICPMS) was applied to quantify Fe and Pt released from FePt-Cys NPs in single HepG2 cells . The developed chip was composed of droplet generation, cell lysis, and droplet-splitting units.…”