MS Identification of Blood Plasma Proteins Concentrated on a Photocrosslinker-Modified Surface

Abstract

This work demonstrates the use of a modified mica to concentrate proteins, which is required for proteomic profiling of blood plasma by mass spectrometry (MS). The surface of mica substrates, which are routinely used in atomic force microscopy (AFM), was modified with a photocrosslinker to allow "irreversible" binding of proteins via covalent bond formation. This modified substrate was called the AFM chip. This study aimed to determine the role of the surface and crosslinker in the efficient concentration of various types of proteins in plasma over a wide concentration range. The substrate surface was modified with a 4-benzoylbenzoic acid N-succinimidyl ester (SuccBB) photocrosslinker, activated by UV irradiation. AFM chips were incubated with plasma samples from a healthy volunteer at various dilution ratios (10²X, 10⁴X, and 10⁶X). Control experiments were performed without UV irradiation to evaluate the contribution of physical protein adsorption to the concentration efficiency. AFM imaging confirmed the presence of protein layers on the chip surface after incubation with the samples. MS analysis of different samples indicated that the proteomic profile of the AFM-visualized layers contained common and unique proteins. In the working series of experiments, 228 proteins were identified on the chip surface for all samples, and 21 proteins were not identified in the control series. In the control series, a total of 220 proteins were identified on the chip surface, seven of which were not found in the working series. In plasma samples at various dilution ratios, a total of 146 proteins were identified without the concentration step, while 17 proteins were not detected in the series using AFM chips. The introduction of a concentration step using AFM chips allowed us to identify more proteins than in plasma samples without this step. We found that AFM chips with a modified surface facilitate the efficient concentration of proteins owing to the adsorption factor and the formation of covalent bonds between the proteins and the chip surface. The results of our study can be applied in the development of highly sensitive analytical systems for determining the complete composition of the plasma proteome.

Keywords: atomic force microscopy; crosslinker; mass spectrometry; protein immobilization.

Figure A1

Typical AFM images of the surface of working experiments after incubation of AFM chips with plasma at an appropriate dilution ratio (left). (right) Cross-section profile corresponding to the lines in the respective AFM images shown on the left: (a) #S.1.1UV+, (b) #S.1.2UV+, (c) #S.1.3UV+.

Figure A2

Typical AFM images obtained in experiments on the estimation of physical adsorption after incubation of the AFM chip with plasma at appropriate dilution ratios. Panels on the right display a cross-section profile corresponding to the lines in the respective AFM images shown on the left: (a) #S.1.1UV−, (b) #S.1.2UV−, (c) #S.1.3UV−.

Figure A3

Typical AFM image of the chip surface after incubation in protein-free solution—PBSD buffer (#S.1.4UV+).

Figure 1

Venn diagrams for MS protein identification. The number of proteins identified on the surface of AFM chips and in plasma samples is presented. Data for dilution ratios of 10²X (a), 10⁴X (b), and 10⁶X (c) are shown. Colors indicate the samples from the working group of AFM chips in series 1. Red: chips incubated with plasma samples upon UV irradiation (designated as #S.1.1UV+, #S.1.2UV+, #S.1.3UV+). Green: samples from the control group of AFM chips in series 1—chips incubated with plasma samples without UV irradiation (designated as #S.1.1UV−, #S.1.2UV−, #S.1.3UV−). Blue: plasma samples included in series 2 for which the protein concentration step was omitted (designated as #P.2.1, #P.2.2, #P.2.3).

Figure 2

MS identification of proteins concentrated on the surface of AFM chips. Scatter plots are presented as log₂-transformed intensity-based absolute quantification (IBAQ) values correlation table. Proteins were detected in eluates from the surface after incubation with diluted plasma samples. For each correlation, Pearson’s correlation coefficient is provided in blue. Numbers 1, 3, and 5 correspond to working AFM chips #S.1.1UV+, #S.1.2UV+, and #S.1.3UV+, respectively. Numbers 2, 4, and 6 correspond to control AFM chips #S.1.1UV−, #S.1.2UV−, #S.1.3UV−, respectively. The X and Y axes correspond to the IBAQ values of proteins identified on the respective AFM chips 1–6.

Figure 3

Venn diagram summarizing the total number of all proteins identified on the surface of the working AFM chips after incubation with plasma samples. Colors indicate the samples from the working group of AFM chips in series_1—chips incubated with plasma samples under irradiation (signatures #S.1.1UV+, #S.1.2UV+, #S.1.3UV+; red); samples from the control group of AFM chips in series_1—chips incubated with plasma samples without irradiation (signatures #S.1.1UV−, #S.1.2UV−, #S.1.3UV−; green); and plasma samples included in series_2, in which no surface protein concentration step was performed (signatures #P.2.1, #P.2.2, #P.2.3; blue).

Figure 4

Volcano plots displaying the MS identification of proteins by plasma analysis at a dilution ratio of 10⁴X with and without the concentration step. (a) Comparison of samples with #S.1.2UV+ (red dots) and #S.1.2UV− (blue dots), (b) Samples with #S.1.2UV+ (red dots) and #P.2.2 (blue dots) are presented. The X-axis shows the log₂ of the fold-change value (log₂(B/A)) of differential protein expression across sample groups, and the Y-axis shows the negative log₁₀ of the p value reflecting changes in gene expression. Each dot in the figure represents a protein. Comparisons between different protein groups are indicated by different colors.

Figure 5

Schematic representation of the processes occurring on the AFM chip surface. Orange letters highlight the functional groups participating in the formation of chemical bonds upon the modification of the substrate surface with the SuccBB crosslinker (orange arrow). Green letters highlight the functional groups participating in forming covalent bonds between the crosslinker-modified AFM chip surface and captured protein upon UV irradiation during the incubation of the chip in the analyzed sample (green arrow).

Figure 6

Schematic of the experiment workflow. The main steps of the experiment are described in the text. The left panel shows the steps related to the work using AFM chips, while the right panel shows the work with plasma samples without concentration on the chip surface.