Quantitation of Intact Proteins in Human Plasma Using Top-Down

Sep 7, 2018 - Abstract | Full Text HTML | PDF w/ Links | Hi-Res PDF · Spectroelectrochemical Nanosensor for the Determination of Cystatin C in Human ...
0 downloads 0 Views 2MB Size
Letter Cite This: Anal. Chem. XXXX, XXX, XXX−XXX

pubs.acs.org/ac

Quantitation of Intact Proteins in Human Plasma Using Top-Down Parallel Reaction Monitoring-MS Yuchao Chen, Pan Mao, and Daojing Wang* Newomics Inc., 804 Heinz Avenue, Suite 150, Berkeley, California 94710, United States

Anal. Chem. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 09/07/18. For personal use only.

S Supporting Information *

ABSTRACT: Direct quantitation of proteins in complex biological matrices by mass spectrometry remains a challenge. Here, we describe a novel top-down parallel reaction monitoring-mass spectrometry (PRM-MS) assay, enabled by microflow LC-nanospray MS using a silicon microfluidic LC-MS chip. We demonstrated direct analysis of intact proteins such as somatropin in human plasma, achieving sensitivity (0.1−1.0 fmole) and speed (1−5 min) on par with enzyme-linked immunosorbent assay (ELISA).

T

simultaneous MS detection and quantitation of multiple fragment ions from each charge state of the protein. Our assay takes advantage of the high sensitivity conferred by the peptide-level MS detection and augmented by PRM, the high specificity conferred by targeted analysis of the intact proteins by high-resolution mass spectrometry (HRMS), and the high speed and high sensitivity of microflow LC-nanospray MS analysis enabled by our integrated silicon microfluidic LC-MS chips. We demonstrated its proof-of-principle application in directly quantifying exogenous somatropin in human plasma within 5 min. We first developed an integrated silicon microfluidic chip (i.e., MEA chip14,15) for rapid LC-MS analysis of intact proteins and peptides (Figure 1a). A LC column was integrated with a multinozzle emitter at the chip level with an internal swept volume of