Methods and materials for assessing and treating cancer

A method for identifying a peptide biomarker, said method comprising:

• digesting polypeptides present in a disease blood sample to obtain disease peptide fragments;
• labeling said disease peptide fragments with a first heavy isotope to obtain labeled disease peptide fragments;
• digesting polypeptides present in a reference blood sample to obtain reference peptide fragments;
• labeling said reference peptide fragments with a second heavy isotope to obtain labeled reference peptide fragments;
• subjecting the labeled disease peptide fragments and the labeled reference peptide fragments to mass spectrometry to identify a peptide biomarker, wherein the level of said peptide biomarker is elevated in the labeled disease peptide fragments relative to the labeled reference peptide fragments.

A method for validating a peptide biomarker, said method comprising:

• subjecting a plurality of peptides comprising said peptide biomarker to basic pH reversed-phase liquid chromatography (bRPLC) to obtain a plurality of fractions;
• organizing said plurality of fractions into a plurality of fraction groups, wherein the number of fractions is higher than the number of fraction groups;
• separating peptide biomarkers in each fraction group by orthogonal high performance liquid chromatography (HPLC) at acidic pH to obtain continuous HPLC elutes;
• and
• analyzing said continuous HPLC elutes using a selected reaction monitoring (SRM) method comprising preoptimized transitions and preoptimized dwell times for said peptide biomarker to determine the intensity of said peptide biomarker;
• wherein the peptide biomarker is validated when the peptide biomarker is detected and quantitated at an elevated level in a disease sample relative to a reference sample using said SRM method.

A method for identifying and validating a peptide biomarker, said method comprising:

• (A) identifying a candidate peptide biomarker, wherein said identifying comprises:
• (i) digesting polypeptides present in a disease blood sample to obtain disease peptide fragments;
• (ii) labeling said disease peptide fragments with a first heavy isotope to obtain labeled disease peptide fragments;
• (iii) digesting polypeptides present in a reference blood sample to obtain reference peptide fragments;
• (iv) labeling said reference peptide fragments with a second heavy isotope to obtain labeled reference peptide fragments;
• (v) subjecting the labeled disease peptide fragments and the labeled reference peptide fragments to mass spectrometry to identify a candidate peptide biomarker, wherein the level of said candidate peptide biomarker is elevated in the labeled disease peptide fragments relative to the labeled reference peptide fragments;
• (B) building a SAFE-SRM method, wherein said building comprises:
• (i) synthesizing said candidate peptide biomarker;
• (ii) subjecting said synthetic candidate peptide biomarker to mass spectrometry to determine a candidate peptide biomarker transition, wherein said transition is determined by identifying a precursor-product ion pair having a strongest intensity and identifying a collision energy (CE) producing said precursor-product ion pair;
• (iii) subjecting a plurality of peptides comprising said candidate peptide biomarker to basic pH reversed-phase liquid chromatography (bRPLC) to obtain a plurality of fractions, wherein said plurality consists of essentially equal amounts of each peptide;
• (iv) organizing said plurality of fractions into a plurality of fraction groups, wherein the number of fractions is higher than the number of fraction groups;
• (v) determining an intensity of said candidate peptide biomarker in each of said fraction groups using the candidate peptide biomarker transition and a fixed dwell time;
• and
• (vi) optimizing the dwell time by re-assembling the transitions according to their hydrophobicity at high pH;
• and
• (C) validating said candidate peptide biomarker, wherein said validating comprises:
• (i) quantitating said candidate peptide biomarker in said disease blood sample, said quantitating comprising:
• (a) subjecting said disease peptide fragments comprising said candidate peptide biomarkers to bRPLC to obtain a plurality of fractions;
• (b) organizing said plurality of fractions into a plurality of fraction groups, wherein the number of fractions is higher than the number of fraction groups;
• (c) separating peptides in each fraction group by orthogonal HPLC at acidic pH to obtain continuous HPLC elutes;
• and
• (d) analyzing said continuous HPLC elutes using a SRM method comprising said candidate peptide biomarker transition and said optimized dwell time;
• (ii) quantitating said candidate peptide marker in said reference blood sample, said quantitating comprising:
• (a) subjecting said reference peptide fragments to bRPLC to obtain a plurality of fractions;
• (b) organizing said plurality of fractions into a plurality of fraction groups, wherein the number of fractions is higher than the number of fraction groups;
• (c) separating peptides in each fraction group by orthogonal HPLC at acidic pH to obtain continuous HPLC elutes;
• (d) analyzing said continuous HPLC elutes using said SRM method comprising said candidate peptide biomarker transition and said optimized dwell time;
• and
• (iii) validating said candidate peptide biomarker when the candidate peptide biomarker is quantitated at an elevated level in said disease sample relative to said reference sample.