Immuno-MALDI-MS in human plasma and on-chip biomarker characterizations at the femtomole level

Abstract

Immuno-SPR-MS is the combination of immuno-sensors in biochip format with mass spectrometry. This association of instrumentation allows the detection and the quantification of proteins of interest by SPR and their molecular characterization by additional MS analysis. However, two major bottlenecks must be overcome for a wide diffusion of the SPR-MS analytical platform: (i) To warrant all the potentialities of MS, an enzymatic digestion step must be developed taking into account the spot formats on the biochip and (ii) the biological relevancy of such an analytical solution requires that biosensing must be performed in complex media. In this study, we developed a procedure for the detection and the characterization at ~1 µg/mL of the LAG3 protein spiked in human plasma. The analytical performances of this new method was established, particularly its specificity (S/N > 9) and sensitivity (100% of LAG3 identification with high significant mascot score >68 at the femtomole level). The collective and automated on-chip MALDI-MS imaging and analysis based on peptidic fragments opens numerous applications in the fields of proteomics and diagnosis.

Appendix 1.

Sequence coverage of the matching peptides detected after reduction and digestion of the deposited RSA at 10 fmol/mm² using 10 and 30 ng/μL of trypsin.

Appendix 2.

sequence coverage (A) and Peptide list (B) obtained after biochemical treatments of the captured LAG3 protein in human plasma related to the green array in Figure 6(A,C).

Figure 1.

Sensorgram of the response of diluted human plasma (2.5%) onto immunochips, especially α-LAG3 (green curve) and α-RSA (red curve) at 25 °C, during 15 min at a flow rate of 20 μL/min.

Figure 2.

Sensorgram of interaction between LAG3 capture in diluted human plasma (2.5%) spiked with LAG3 (12.5 nM) with α-LAG3 (green curve) and α-RSA (in red) biochip surface. Differential of the response was indicated by the grey curve.

Figure 3.

on chip MALDI-MS imaging and analysis of a BSA digest after matrix deposition by Imageprep. (A) The distribution of a specific tryptic BSA peptide (m/z 1487.79) is shown in red. (B) MS spectrum obtained on one laser position in the 5 fmol BSA digest spot identifying Bovin Serum Albumin with a Mascot Score of 123.00 and 13 specific peptides (C).

Figure 4.

Mascot Identification score obtained and the number of peptides detected after reduction and digestion of the deposited RSA at 10 fmol/mm² using 10 or 30 ng/μL of trypsin (A). MS spectrum of RSA after digestion by the solution containing 30 ng/μL of trypsin (B) Difference in intensity of a specific RSA peak (1,960.05 m/z) between 10 ng/μL (blue spectrum) and 30 ng/μL (red spectrum) conditions (C).

Figure 5.

Peptide distribution 2211.10 (specific to trypsin), 1960.05 and 1439.78 (specific to RSA), after analysis of a 10 fmol/mm² RSA layer digested by trypsin at 30ng/μl with the deposition of HCCA matrix at 1 mg/mL.

Figure 6.

(A) Sensorgram of LAG3 capture in diluted total human plasma (2.5%) on α-LAG3 (in green, red and blue) and α-RSA (in pink). (B) On-chip MALDI-MS image showing the distribution of the specific peptides from LAG3 (m/z 1,422.69) in red, α-RSA (m/z 1,697.20) in yellow and calibration sample (Pepmix, m/z 1,296.68) in green. (C) Results of on-chip MALDI-MS and MS/MS analysis. (D) MS/MS spectrum obtained for the peak m/z 1,422.69. (E) On-chip MALDI-MS spectrum of LAG3 after in situ tryptic digestion.