High-Resolution Analysis of Antibodies to Post-Translational Modifications Using Peptide Nanosensor Microarrays - PubMed Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Dec 27;10(12):10652-10660.
doi: 10.1021/acsnano.6b03786. Epub 2016 Sep 20.

High-Resolution Analysis of Antibodies to Post-Translational Modifications Using Peptide Nanosensor Microarrays

Jung-Rok Lee, D James Haddon, Nidhi Gupta  1 , Jordan V Price  2 , Grace M Credo  1 , Vivian K Diep, Kyunglok Kim, Drew A Hall  3 , Emily C Baechler  4 , Michelle Petri  5 , Madoo Varma  1 , Paul J Utz  6 , Shan X Wang
Affiliations

High-Resolution Analysis of Antibodies to Post-Translational Modifications Using Peptide Nanosensor Microarrays

Jung-Rok Lee et al. ACS Nano. .

Abstract

Autoantibodies are a hallmark of autoimmune diseases such as lupus and have the potential to be used as biomarkers for diverse diseases, including immunodeficiency, infectious disease, and cancer. More precise detection of antibodies to specific targets is needed to improve diagnosis of such diseases. Here, we report the development of reusable peptide microarrays, based on giant magnetoresistive (GMR) nanosensors optimized for sensitively detecting magnetic nanoparticle labels, for the detection of antibodies with a resolution of a single post-translationally modified amino acid. We have also developed a chemical regeneration scheme to perform multiplex assays with a high level of reproducibility, resulting in greatly reduced experimental costs. In addition, we show that peptides synthesized directly on the nanosensors are approximately two times more sensitive than directly spotted peptides. Reusable peptide nanosensor microarrays enable precise detection of autoantibodies with high resolution and sensitivity and show promise for investigating antibody-mediated immune responses to autoantigens, vaccines, and pathogen-derived antigens as well as other fundamental peptide-protein interactions.

Keywords: autoantibody; giant magnetoresistance; lupus; nanosensors; peptide microarray; regeneration.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Development and validation of GMR nanosensor peptide microarrays. (a) List of peptides used in GMR nanosensor microarray. Acetylated lysine (K*) and alanine substitution (A) are represented in blue and red, respectively. (b) Schematic of GMR nanosensor peptide microarray (not to scale). A panel of peptides (indicated by different colors) was spotted on a GMR nanosensor chip. Antibody-containing samples were used to probe the microarrays, combined with biotinylated secondary antibodies (illustrated with purple tips) and streptavidin-coated MNPs. The stray field from the bound MNPs was detected by the nanosensors underneath. Inset: 16 sensors from an 8 × 8 GMR nanosensor chip (peptides were spotted on 3 sensors in the second row from the top). (c) Real-time signals obtained from the GMR nanosensor peptide microarray probed with anti-FLAG and anti-K5Ac antibodies. MNPs were added to the microarray at ~1.5 min as indicated by the dotted line. (d) Titration curves of anti-FLAG and anti-K5Ac antibodies measured with the microarrays. Error bars represent standard deviations of 4 identical sensor signals.
Figure 2
Figure 2
GMR nanosensor peptide microarrays identify IgG autoantibodies to H2B in the sera of individuals with SLE. Sera from two H2B positive individuals with SLE (SLE41 and SLE47) and two healthy controls (HC1 and 2) were measured with the microarrays. Buffer solution without sera was used as a negative control (NC). The signal intensity of each peptide feature is the average of 4 identical sensor signals.
Figure 3
Figure 3
(a) Real-time signals obtained during the probing and regeneration. Anti-FLAG (1 µg/mL), anti-K5Ac (0.5 µg/mL), and anti-H2B (5 µg/mL) antibodies were incubated with the microarray. The MNPs were added at ~3 min, and the signals reached their plateaus at ~20 min. Glycine-HCl pH 2.0 was added to the microarray at ~23 min, as indicated by the arrow. Bovine serum albumin (BSA) and peptide AIYAAPFK were used as negative controls. Error bars represent standard deviations of 4 identical sensor signals. (b) Residual signals for each peptide-antibody pair were calculated after 20 min of regeneration with 5 different regeneration solutions. Residual signals were calculated with respect to their plateau signals. (c) Residual signals for each peptide–antibody complex were monitored for 30 min after regeneration solutions were added. All signals were referenced to BSA signals.
Figure 4
Figure 4
(a) Reproducibility of measurements with regeneration. A sample containing anti-FLAG (1 µg/mL), anti-K5Ac (0.5 µg/mL), and anti-H2B (5 µg/mL) antibodies was measured before and after 1 h of regeneration with 5 different regeneration solutions. The reproducibility is shown as the signals obtained in the second run as a percentage of those in the first run for each peptide–antibody complex (blue: FLAG–anti-FLAG, red: H2B 2–21 AllAc–anti-K5Ac, and green: H2B 1–5–anti-H2B). (b) Five cycles of probing with the same antibody mixture, followed by 1 h regeneration with glycine-HCl pH 2.0 were performed with a single chip. “S” indicates the plateau signals from the sample measurement, and “R” represents the signals at the end of regeneration. After each regeneration, the chip was neutralized with 1% BSA for 30 min. All signals were referenced to BSA signals. Error bars represent standard deviations of 4 identical sensor signals.
Figure 5
Figure 5
(a) Schematic of peptide synthesis directly on GMR nanosensor microarray using photolithography. After a seed layer is created on GMR nanosensors (blue, red, and green rectangles), a photoacid-generating photoresist layer is coated on the surface. The red diamonds represent t-BOC groups (tert-butyloxycarbonyl group), and the circles with different colors are amino acids. Maskless DMD technology is used to selectively expose the photoresist to UV light, deprotecting the t-BOC end groups via generation of photoacid. The deprotected amino acids are coupled with the next amino acids. (b) FLAG (blue), K3A mutant FLAG (FLAG Mut, red), and FLAG without the M2 epitope (FLAG Del, green) were synthesized on the microarrays and used to measure anti-FLAG antibodies at 1 µg/mL. (c) In situ synthesized and spotted peptide microarrays featuring four peptides (H2B 1–20 AllAc, H2B 1–20 Mut, H2B 2–21 AllAc, and H2B 2–21 Mut) were probed with anti-K5Ac antibodies at 0.2 µg/mL. (d) Three cycles of measurements and regeneration (glycine-HCl pH 2.0) were performed with an in situ synthesized GMR nanosensor peptide microarray. Cycle 1: anti-K5Ac antibodies at 0.2 µg/mL (blue bars). Cycles 2 and 3: anti-FLAG (0.5 µg/mL) and anti-K5Ac (0.2 µg/mL) antibodies (cycle 2: red, and cycle 3: green).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Obermoser G, Pascual V. The Interferon-Alpha Signature of Systemic Lupus Erythematosus. Lupus. 2010;19:1012–1019. - PMC - PubMed
    1. Yaniv G, Twig G, Shor DB, Furer A, Sherer Y, Mozes O, Komisar O, Slonimsky E, Kiang E, Lotan E, Welt M, Maraj I, Shina A, Amital H, Shoenfeld Y. A Volcanic Explosion of Autoantibodies in Systemic Lupus Erythematosus: a Diversity of 180 Different Antibodies Found in SLE Patients. Autoimmun. Rev. 2015;14:75–79. - PubMed
    1. van Boekel MA, Vossenaar ER, van den Hoogen FH, van Venrooij WJ. Autoantibody Systems in Rheumatoid Arthritis: Specificity, Sensitivity and Diagnostic Value. Arthritis Res. 2002;4:87–93. - PMC - PubMed
    1. Arbuckle MR, McClain MT, Rubertone MV, Scofield RH, Dennis GJ, James JA, Harley JB. Development of Autoantibodies before the Clinical Onset of Systemic Lupus Erythematosus. N. Engl. J. Med. 2003;349:1526–1533. - PubMed
    1. Nielen MM, van Schaardenburg D, Reesink HW, van de Stadt RJ, van der Horst-Bruinsma IE, de Koning MH, Habibuw MR, Vandenbroucke JP, Dijkmans BA. Specific Autoantibodies Precede the Symptoms of Rheumatoid Arthritis: a Study of Serial Measurements in Blood Donors. Arthritis Rheum. 2004;50:380–386. - PubMed

Publication types