Systems and methods for assaying large molecules with improved sensitivity
AssigneeGenentech, Inc.
InventorsKeyang Xu, Jintang He, Surinder Kaur
Life of this patent
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Abstract
The present application relates to systems and methods for assaying presence of large molecule analytes, such as proteins, e.g., antibodies, antigens, receptors, and the like, using a targeted two-dimensional liquid chromatography, tandem mass spectrometry (2D-LC-MS/MS) system, optionally combined with affinity capture. In some aspects, the system is partially or fully automated. In some aspects, the system may allow detection of protein biomarkers (e.g., antibodies or antigens) from clinical or nonclinical biological tissue or fluid samples in the pg/mL to ng/mL range.
Claims19
A method of detecting an analyte in a sample, comprising:
- (a) performing high pH reversed phase liquid chromatography (RPLC) survey scan with detection by mass spectrometry (MS) on the analyte at previously determined concentration in the presence of an internal standard, to determine the peak apex (Tapex) of the analyte;
- (b) obtaining an experimental sample comprising the analyte;
- (c) performing high pH RPLC on the sample of (b);
- (d) collecting at least a portion of the high pH RPLC eluate of (c) corresponding to Tapex for the analyte+/−average peak width of the analyte or of the internal standard in the survey scan of (a), wherein the at least a portion is optionally collected in a trap column;
- (e) performing low pH RPLC on the collected portion of the high pH RPLC eluate of (d);
- and
- (f) detecting the analyte by MS.
A method of detecting a peptide analyte in a sample, comprising:
- (a) obtaining an experimental sample and performing an affinity-based capture to isolate a protein in the sample;
- (b) digesting the captured protein with a protease to obtain a peptide analyte for detection;
- (c) performing high pH reversed phase liquid chromatography (RPLC) survey scan with detection by mass spectrometry (MS) on the peptide analyte at previously determined concentration in the presence of an internal standard peptide, to determine the peak apex (Tapex) of the analyte;
- (d) performing high pH RPLC on the digested sample of (b) comprising the peptide analyte;
- (e) collecting at least a portion of the high pH RPLC eluate of (d) corresponding to Tapex for the analyte+/−average peak width of the analyte, the internal standard, or a peptide in the survey scan of (c), wherein the at least a portion is optionally collected in a trap column;
- (f) performing low pH RPLC on the collected portion of the high pH RPLC eluate of (e);
- and
- (g) detecting the analyte by MS.
Dependencies are inferred from the claim text.