WO2023089533A1

WO2023089533A1 - Tandem triple quadrupole mass spectrometer-based screening and confirmation method for analyzing protein and its variants

Info

Publication number: WO2023089533A1
Application number: PCT/IB2022/061103
Authority: WO
Inventors: Deepalakshmi PD
Original assignee: Neuberg Anand Academy Of Laboratory Medicine Private Limited
Priority date: 2021-11-18
Filing date: 2022-11-17
Publication date: 2023-05-25

Abstract

The present disclosure relates to a system and method for screening and confirming protein and protein variants in tandem triple quadrupole mass spectrometer (TQMS) using pseudo–Parallel Reaction Monitoring (pPRM) method. More particularly, the invention provides complete peptide spectra, comprising all the amino acids of the peptide, in a single analysis. In additional embodiments, the invention deals with the Pseudo Parallel Reaction Monitoring (pPRM) method using Electrospray Ionization Triple Quadrupole Mass Spectrometer (ESI TQMS). The method is a QUALITATIVE test to CONFIRM the PRESENCE of the protein and its variants of interest and can be used with or without a separating device like liquid chromatography. This is achieved by monitoring the product ions (>5 ions) OR ALL THE PRODUCT IONS ('y' ion series) of the peptide.

Description

TANDEM TRIPLE QUADRUPOLE MASS SPECTROMETER-BASEDSCREENING AND CONFIRM TION METHOD FOR ANALYZING PROTEIN AND ITS VARIANTS

TECHNICAL FIELD OF THE INVENTION

[01] The present invention is in the technical field of a system and a method for screening protein and protein variants using pseudo-Parallel Reaction Monitoring (pPRM) method. More particularly, Invention provides complete peptide spectra, comprising all the amino acids, in a single analysis.

BACKGROUND ART

[02] Triple quadrupole mass spectrometry (TQMS) is used in multiple industries like pharmaceutical, food, environmental, forensics, clinical labs, and quality control laboratories. These methods are mainly used as a quantitative tool to identify and estimate relative and/or absolute quantities of organic compounds in a sample. In a tandem triple quadrupole MS instrument, a specific substance (precursor ion) is selected in the first quadrupole, which collides with an inert gas in the secondquadrupole, to generate characteristic fragment ions or product ions.

[03] These product ions are selectively monitored by the third quadrupole. The combined monitoring of a precursor ion and its characteristic product ion is termed asmass transition. The signal intensity of a specific transition is directly related to the concentration of analyte in the sample. If one or more transitions (1-5) are monitored after the reaction in a collision cell (second quadrupole), it is termed single or multiplereaction monitoring (SRM/MRM).

[04] Developing an MRM method needs elaborate optimizations to generateidealized assays in a TQMS (A Doerr, 2012). Although monitoring more mass transitions increases the selectivity and specificity of molecules, sensitivity is compromised.

[05] Moreover, when molecules with higher masses (>800Da) like peptides are characterized in a TQMS, a maximum of 2-5 product ions are monitored for quantifying the peptide in a sample. In a complex mixture of proteins as in a clinical sample, hundreds of peptides are generated by digesting the proteins with enzymes like trypsin, which cleaves specifically to the C terminal of arginine and lysine residues(Olsen IV, Ong SE, Mann M, 2004).

[06] These tryptically digested complex biological samples have interfering ions (isobaric compounds that have the same nominal mass but different molecular structure) which cannot be eliminated in a triple quad due to their low resolution (Gallien S, Duriez E, Demeure K, 2013). Moreover, the mass similarities of different amino acids that make up the peptide reduce the selectivity of the peptide. For eg., a mass difference between two spectral peaks of 114Da indicates the presence of asparagine. The mass also corresponds to two glycine molecules. Monitoring only 2- 5 transitions for a peptide is, therefore, not specific to the peptide.

[07] Therefore, all transitions of a peptide are to be monitored for confirmation of the proteins present in a sample. This is currently available only on a high-resolution mass spectrometry (HRMS), where all the ions are monitored parallelly with high mass accuracy (parallel reaction monitoring - PRM) to confirm the sequence of the peptideand hence the presence of the protein (Mesri, 2014).

[08] Comparison of SRM on a TQMS and PRM on a HRMS is largely focused onthe quantitation of proteins in a targeted approach (Mesri, 2014; Peterson et al., 2012;Ronsein et al., 2015).

[09] In the present scenario, confirmation for the presence of the protein is done by using HRMS by PRM method or by molecular sequencing techniques. The majority of the protein applications using ESI TQMS are to QUANTIFY the PEPTIDES by monitoring 2-5 PRODUCT IONS. The selectivity of the peptide is compromised for confirming the presence of the protein when only 2-5 product ions are monitored. However, such methods have lots of drawbacks.

[10] Current MS technology to confirm the presence of a peptide largely is based on a quadrupole-equipped high-resolution mass spectrometer, which allows parallel detection of all the transitions in a single analysis, termed parallel reaction monitoring (PRM) (A C Peterson, J D Russell, D J Bailey, 2012). The optimizations that are required to achieve this in a high-resolution MS, require less effort when compared totriple quad instruments.

[11] Moreover, the presence of interfering ions in a full mass spectrum is less troublesome to the overall quality of the spectrum than the presence of interfering ions in a narrow mass range, because many ions would be available for analysis. However, the cost of equipment and maintenance is high and requires an expert to operate the machine and interpret the data.

[12] A different type of mass spectrometer (MALD1 Matrix Assisted Laser Desorption Ionization Mass Spectrometry) was successfully implemented in the clinical laboratories for qualitative analysis of microorganisms with an in-built proteindatabase, in an in vitro diagnostic mode (IVD)(A. Bizzini, 2010). Confirmation of the peptide sequence is not possible in IVD mode due to the lack of fragmentation optionsin the system and the poor resolution of the instrument.

[13] TQMS is widely used in clinical laboratories around the world, for the quantitation of metabolites - drug metabolites (H. Schupke, R. Hempel, R. Eckardt, 1996; R. Kostiainen, T. Kotiaho, T. Kuuranne, 2003) hormones (S.S.-C. Tai, D. M. Bunk, E.White V, 2004)), vitamins (Chatzimichalakis et al., 2004) with great precisionbut has been recently adapted for quantitation of protein and peptide measurements (Ronsein et al., 2015). Many TQMS methods are available for population screening ofHb disorders, particularly HbS based on the intact mass difference between wild typeand the variant (B. J. Wild, B.N. Green, E.K.Cooper, M.R.A.Lalloz, S.Erten, A. D. Stephens, 2001; Raymond Neil Dalton and Charles Turner, 2004). Since intact mass is not specific enough to identify the defective protein, proteins were digested with trypsin and the resulting peptides were monitored for the presence of variant peptides (Y. A. Daniel et al., 2005a) either in whole blood or in dried blood spots (Charles T, Raymond N D, 2014; Y. Daniel & Turner, 2018). Another variant of the Mass Spectrometer utilizes an ion trap to determine the sequence of Hb peptides (Haynes et al., 2013). Due to its poor sensitivity and dynamic range, its usage is limited in clinical laboratories. Chromatographic techniques like high-pressure liquid chromatography (HPLC) (Joutovsky A, Hadzi- Nesic J, 2004), capillary electrophoresis (Mario N, Baudin B, Bruneel A, Janssens J, 1999), and low- resolution mass spectrometers (Wild et al., 2004) have been demonstrated for high-throughput population screening of the clinically important hemoglobinopathies (Y. A. Daniel etal., 2005b).

[14] Molecular techniques (Tan AS, Quah TC, Low PS, 2001) confirm the presence of the defective gene by sequencing the gene or specific region of the gene of interest. Molecular testing is not cost-effective for routine analysis and is not done in all diagnostic laboratories. Currently, it is the only technique to confirm the presenceof the variant in a clinical sample.

[15] At present, in the clinical diagnostic industry and many other analytical laboratories, TQMS is the gold standard for the quantitation of molecules (H. Schupke,

R. Hempel, R. Eckardt, 1996)’(R. Kostiainen, T. Kotiaho, T. Kuuranne, 2003) but not for qualitative analysis. A maximum of 3-5 product ions are monitored which improves the specificity of the protein being identified (H. Keshishian, T. Addona, M. Burgess, E. Kuhn, 2007), but still not sufficient for confirmation of the proteins presentin the sample, unlike PRM assays on HRMS.

[16] Moreover, PRM assay on HRMS is not affordable by all and still not widely used in clinical and analytical laboratories for routine analysis.

[17] In summary, there is a need in the art for a robust, cost-effective, and selective method for clinical laboratories for screening and confirmation method for the presence of protein and its variants.

[18] SUMMARY OF THE INVENTION

[19] The primary objective of the present invention is to provide a pseudoPRM method based complete peptide sequence, comprising all the transitions of a peptide in MRM mode, in a single analysis in a TQMS.

[20] In an embodiment, it is called pseudoPRM, because the quadrupole scans each ion, one at a time and at unit resolution as opposed to the parallel monitoring of all product ions of a peptide with high mass accuracy in HRMS .

[21] In a further embodiment, the invention provides confident identification and confirmation of the peptide sequence and therefore the protein present in a sample.

[22] In an embodiment, the selectivity and specificity are greatly enhanced due tothe monitoring of all the transitions of a peptide in a single analysis.

[23] In an exemplary embodiment, the invention deals with Pseudo Parallel Reaction Monitoring (pPRM) method using ESI TQMS:

[24] It is a QUALITATIVE test to CONFIRM the PRESENCE of the protein of interest. This is achieved by monitoring greater than 5 OR ALL THE PRODUCT IONS (‘y’ ion series) of the peptide.

[25] Hemoglobin is used as a model protein to demonstrate the new method.

[26] Several aspects of the invention are described below with reference to examples for illustration. However, one skilled in the relevant art will recognize that the invention can be practiced without one or more of the specific details or with othermethods, components, materials, and so forth. In other instances, well-known structures, materials, or operations are not shown in detail to avoid obscuring the features of the invention. Furthermore, the features/aspects described can be practiced in various combinations, though only some of the combinations are described herein for conciseness.

BRIEF DESCRIPTION OF THE DRAWINGS

[27] Example embodiments of the present invention will be described with reference to the accompanying drawings briefly described below.

[28] FIG. 1 illustrates the schematic representation of PRM (A) in a QqOrbitrap (HRMS) and pPRM (B) in a triple quad QqQ according to the aspects of the invention. In PRM, all product ions are monitored simultaneously, which correctly identifies thepeptide from a complex biological sample, whereas in pPRM, product ions of the peptide, sufficient to confirm the sequence of the peptide is monitored, according to the aspects of the invention

[29] FIG. 2A-D illustrate the PseudoPRM of a tryptically digested Hb protein resulting in peptides BT1(A), BT3(B), BT12(C) (from 0 chain of HbAO standard), and STl(D) peptide of HbS standard. FIG.2A illustrates Pseudo PRM spectra of tryptic peptide 1 of 0 chain of Hb [M+2H]2+, 476.8m/z from Hb standard HbAO. All the y ion fragment series (y l-y7) and b ion fragment series (b2-b4, b6) were observed confirming the presence of the 0 chain of Hb. FIG.2B illustrates Pseudo PRM spectraof tryptic peptide 3 of 0 chain of Hb [M+2H]2+, 657.8 m/z from Hb standard HbAO. All the y ion series fragments (yl-yl2) provide a complete sequence of the peptide. The b ion series fragments (b2-b5) provide additional confirmation of the peptide at the N terminal region confirming the presence of the 0 chain of Hb. FIG.2C illustrates Pseudo PRM spectra of tryptic peptide 12 of 0 chain of Hb [M+2H]2+, 689.8 m/z of Hb standard HbAO. All the y ion fragment series (yl-y 11) and intense b2 and b3 diagnostic ions confirm the presence of the 0 chain of Hb. FIG. 2D illustrates Pseudo PRM spectra of tryptic peptide STI of Hb [M+2H]2+, 461.8 m/z of HbS standard. Observation of the y ion series (yl-y7) indicates the complete sequence information of the variant peptide. The b ion series fragments (b2, b3) provide additional confirmation of the peptide at the N terminal region thereby confirming the presence of HbS, according to the aspect of present invention.

[30] FIG. 3A-E illustrate the PseudoPRM spectra of the synthesized peptides representing Hb variants HbS(A), HbC(B), HbE(C), HbD(D) and HbO(E) according to the aspects of the invention. FIG. 3A illustrates Pseudo PRM spectra of synthetic HbS peptide of Hb [M+2H]2+, 461.8 m/z. Optimization of cone voltage and collision energies to yield all product ions of HbS peptide using synthetic HbS. FIG. 3B illustrates Pseudo PRM spectra of synthetic HbC peptide of Hb [M+2H]²⁺, 347.7 m/z. Optimization of cone voltage and collision energies to yield all product ions of HbC peptide using synthetic HbC. Since it is a small peptide of 6 amino acids, both ion types b and y were observed to improve confident identification. yl-y5 and b2-b4 ions are observed which provides information of the sequence of the peptide in both directions N-C terminal and C-N terminal. FIG. 3C illustrates Pseudo PRM spectra of synthetic HbE peptide of Hb [M+2H]²⁺, 458.7 m/z. Optimization of cone voltage andcollision energies to yield all product ions of HbE peptide using synthetic HbE. FIG. 3D illustrates Pseudo PRM spectra of synthetic HbD peptide of Hb [M+2H]²⁺, 689.4 m/z. Optimization of cone voltage and collision energies to yield all product ions of HbD peptide using synthetic HbD. FIG. 3E illustrates Pseudo PRM spectra of synthetic HbO peptide of Hb [M+2H]²⁺, 625.3 m/z. Optimization of cone voltage andcollision energies to yield all product ions of HbO peptide using synthetic HbO. y5-y7 ions have been magnified 5 times to reveal the ions, according to the aspect of present invention.

[31] FIG. 4A-4D illustrate Pseudo PRM spectra of tryptic peptide HbS, HbC, HbE, and HbD obtained from whole blood of adult patients according to the aspects of the invention. FIG. 4A illustrates Pseudo PRM spectra of tryptic peptide STI of HbS [M+2H]²⁺, 461.8 m/z. Observation of the y ion series (yl-y7) indicates the complete sequence information of the variant peptide thereby confirming the presenceof HbS in a patient’s sample. FIG.4B illustrates Pseudo PRM spectra of tryptic peptideof HbC [M+2H]²⁺, 347.7 m/z. Observation of the y ion series (yl-y5 and b2-b4) indicates the complete sequence information of the variant peptide thereby confirming the presence of HbC in a patient’s sample. FIG. 4C illustrates Pseudo PRM spectra of the tryptic peptide of HbE [M+2H]²⁺, 458.7 m/z. Observation of y ion series (yl-y8) and b2 ion indicates the complete sequence information of the variant peptide thereby confirming the presence of HbE in a patient’s sample. FIG. 4D illustrates Pseudo PRMspectra of the tryptic peptide of HbD [M+2H]²⁺, 689.4 m/z. b2 and b3 ions are the diagnostic ions since the mutation is at the N terminal amino acid of the peptide. Together with the y ion series (yl-yll) the complete sequence information of the

variant peptide is identified thereby confirming the presence of HbD in a patient’s sample, according to the aspect of present invention.

[32] FIG. 5 illustrates NewBom Screening_Dried Blood Spot patient samples comprising Hb variants HbS, HbC, HbE, HbD according to the aspects of the invention. Pseudo PRM spectra of the Hb variants HbS, HbC, HbE, HbD from dried blood spots (DBS) of newborns (4 different patient samples). Observation of the complete sequence of the peptides confirms the presence of these variants in the patient’ s sample, according to the aspect of present invention.

[33] FIG. 6 illustrates electrospray ionization mass spectrometry (ESI-MS) of Delta tryptic peptide 2 (8T2) of 5 chain of Hb confirming Hb Lepore variant according to the aspects of the invention. Pseudo PRM spectra of tryptic peptide δT2 of δ chain of Hb [M+2H]²⁺, 480.3 m/z. Observation of y ion fragment series (yl-y8) confirms the presence of Hb Lepore in the whole blood of a patient. The presence of Hb Lepore, a 80 fusion protein, is confirmed when complete sequence of δT2 peptide is observed, according to the aspect of present invention.

[34] FIG. 7 illustrates Modified peptide (Glycated Hb) according to the aspects ofthe invention. Pseudo PRM spectra of the glycated peptide at the N terminal of p chainof Hb [M+2H]2+, 429.2 m/z. The modified Hb on digestion with GluC, yields N terminal of p chain of Hb [M+2H]2+, 429.2 m/z. In addition to diagnostic ions 110.1m/z and 220.2 m/z, the additional y ion fragment series confirms the presence ofthe modified glycated Hb at the N terminal region of the β chain of Hb, according to the aspect of present invention.

[35] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding referencenumber.

DETAILED DESCRIPTION OF THE INVENTION

[36] It is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forthin the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or of being carried out in variousways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including”, “comprising” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms “a” and “an” herein do not denote a limitation of quantity, but ratherdenote the presence of at least one of the referenced items. Further, the use of terms “first”, “second”, and “third”, and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.

[37] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. By way of example, “a dosage” refers to one or more than one dosage.

[38] The terms “comprising”, “comprises” and “comprised of’ as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elementsor method steps.

[39] All documents cited in the present specification are hereby incorporated by reference in their totality. In particular, the teachings of all documents herein specifically referred to are incorporated by reference.

[40] Example embodiments of the present invention are described with reference to the accompanying figures.

[41] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding referencenumber.

[42] DEFINITIONS

[43] “sample” refers to any sample of a biological nature that requires analysis. For example, samples may include proteins or peptides extracted from tissues, body fluids, cells of an animal, plant, fungus, bacteria or other microorganisms. It can also include peptides that are chemically synthesised.

[44] "protein variant" refers to a member of a set of highly similar proteins that originate from a single gene or gene family and are the result of genetic differences.

[45] “Electrospray ionization” refers to an Electrospray ionization (ESI) techniqueto generate ions for mass spectrometry using electrospray by applying a high voltage to a liquid to produce an aerosol. [46] “Pseudo-Parallel reaction monitoring (PRM)” refers to an ion monitoring technique based on low resolution mass spectrometry.

[47] “Quadrupole” refers to a component of the mass spectrometer responsible for selecting sample ions based on their mass-to-charge ratio (m/z).

[48] “mass spectrum” refers to the m/z ratios of the ions present in a sample plotted against their intensities.

[49] “Synthetic peptide” refers to peptides that are synthesized instead of using phage or biological systems.

[50] “collision cell” refers to a device used in mass spectrometry to remove interfering ions through ion/neutral reactions and to provide structural information ofthe molecules.

[51] “triple quadrupole mass spectrometer (TQMS)” refers to a tandem mass spectrometer made up of two quadrupole mass analyzers, with a (non-mass-resolving) radio frequency-only quadrupole between them, acting as a collision cell for collision- induced dissociation (CID) to fragment the selected precursors ions, and to generate fragment ions.

[52] “LC-MS” refers to an analytical technique that involves physical separation of target compounds (or analytes) followed by their mass-based detection.

A. EMBODIMENTS OF THE INVENTION:

[53] According to a first aspect, a method for screening / analyzing ! confirming aprotein variant in a sample is provided. The method includes the steps of: (i) processinga protein to produce a series of peptides; (ii) ionizing the series of peptides in a source region of Electrospray ionization Tandem Triple Quad Mass Spectrometer; (iii) selecting proteotypic peptide ion specific to the protein variant in a first quadrupole and fragmenting the selected proteotypic peptide ion in a collision cell; (iv) selecting greater than five product ions representative of the proteotypic peptide ion in pseudoParallel Reaction Monitoring mode by a third quadrupole; (v) monitoring the selectedproduct ions by a detector; and (vi) detecting the presence of the peptide using the Electrospray ionization Tandem Triple Quad Mass Spectrometer, wherein a mass/charge ratio (m/z) of the product ions and the proteotypic peptide ion indicates the presence of the protein variant in the sample. [54] In an embodiment, the method includes visualizing the mass spectra obtained by the Electrospray ionization Tandem Triple Quad Mass Spectrometer, wherein the mass spectra are a direct readout of the sequence of the peptide.

[55] In yet another embodiment, the quadrupole scans all proteotypic peptide ions at unit resolution with a scan time of 0.1-0.005 seconds.

[56] In yet another embodiment, the protein variant is a hemoglobin variant, insulin, Immunoglobulins, MTb peptides, and membrane proteins.

[57] In yet another embodiment, the hemoglobin variant HbS, HbC, HbE, HbD, HbO, Hb Lepore, Modified glycated peptide of Hb.

[58] In yet another embodiment, the protein is processed by digestion, wherein the protein is digested with N-tosyl-L-phenylalanine chloromethyl ketone treated trypsin.

[59] In yet another embodiment, the sample is a biological sample, wherein the biological sample is whole blood, Dried Blood spot (DBS), whole blood spotted onto Dried blood spot (DBS), urinary protein, Cerebro spinal fluids, and body fluids.

[60] In yet another embodiment, the sample is a plant protein or an animal protein.

[61] According to the second aspect, a method for screening / analyzing / confirming a protein variant in a sample is provided. The method includes the steps of: (i) processing a protein to produce a series of peptides; (ii) subjecting the series of peptides into a liquid chromatography reverse phase column; (iii) separating the peptides in the liquid chromatography reverse phase column under gradient conditions; (iv) ionizing the separated peptides in a source region of Electrospray ionization Tandem Triple Quad Mass Spectrometer; (v) selecting proteotypic peptideion specific to the protein variant in a first quadrupole and fragmenting the selected proteotypic peptide ion in a collision cell; (vi) selecting greater than five product ions representative of the proteotypic peptide ion in pseudoParallel Reaction Monitoring mode by a third quadrupole; (vii) monitoring the selected product ions by a detector; and (viii) detecting the presence of the peptide using the Electrospray ionization Tandem Triple Quad Mass Spectrometer, wherein a mass/charge ratio (m/z) of the product ion and the proteotypic peptide ions eluting at a specific retention time (RT) indicates the presence of the protein variant in the sample. [62] In an embodiment, the method includes visualizing the mass spectra obtained by the Electrospray ionization Tandem Triple Quad Mass Spectrometer, wherein the mass spectra are a direct readout of the sequence of the peptide.

[63] In another embodiment, the method includes using synthetic peptides to optimize peptide fragmentation patterns.

[64] In yet another embodiment, the synthetic peptides are diluted in Liquid Chromatography Mass Spectrometer solvent without tryptic digestion to optimize ionization of the peptides.

[65] In yet another embodiment, the Liquid Chromatography Mass Spectrometer solvent is acidified acetonitrile.

[66] In yet another embodiment, the gradient conditions are between 20-40% Acetonitrile, <0.5% Formic acid in 1-3.5 mins.

[67] In yet another embodiment, the specific retention time (RT) comprises between 1.0-3.5 minutes depending on the peptide variant.

[68] In yet another embodiment, the liquid chromatography reverse phase column is an ultrapressure liquid chromatography reverse phase column. The Liquid Chromatography column may be a High-performance liquid chromatography, a micro- liquid chromatography column, nano-liquid chromatography column. The conditions may vary for each type of the liquid chromatography column and also depend on the complexity of the sample, whether it is a pure protein or present in a simple mixture or in a complex mixture (plasma or serum matrix). The series of peptides may be injected at a Flow rate of 0.1-1.0 ml/minute in the liquid chromatography reverse phasecolumn. The flow rate for the liquid chromatography column is 100s of microliter/minute. The liquid chromatography column may be a micro-liquid chromatography column with a flow rate of 10s of microliter/minute or a nano-liquidchromatography column with a flow rate of nanoliter/minute.

[69] In yet another embodiment, the quadrupole scans all proteotypic peptide ions at unit resolution with a scan time of 0.1-0.005 seconds.

[70] In yet another embodiment, the protein variant is a hemoglobin variant, insulin, Immunoglobulins, MTb peptides, and membrane proteins. [71] In yet another embodiment, the hemoglobin variant is HbS, HbC, HbE, HbD, HbO, Hb Lepore, Modified glycated peptide of Hb.

[72] In yet another embodiment, the protein is processed by digestion, wherein the protein is digested with N-tosyl-L-phenylalanine chloromethyl ketone treated trypsin and diluted in the Liquid Chromatography Mass Spectrometer (LCMS) solvent. Proteins with cysteine bonds are subjected to reduction and alkylation before subjecting to LCMS.

[73] In yet another embodiment, the sample is a biological sample, wherein the biological sample is whole blood, Dried Blood spot (DBS), whole blood spotted onto DBS, urinary protein, Cerebro spinal fluids, and body fluids.

[74] In yet another embodiment, the sample is a plant protein or an animal protein.

[75] According to the aspects of the invention, it provides a complete peptide sequence, comprising all the transitions of a peptide in MRM mode, in a single analysis in a TQMS . The said method is called pseudoPRM, because the quadrupole scans each ion, one at a time and at unit resolution as opposed to the parallel monitoring of all product ions of a peptide with high mass accuracy in HRMS.

[76] In further embodiments, the said method is designed for qualitative analysis, wherein the laborious optimizations of collision energies and cone voltages forindividual transitions required to generate an MRM quantitative method are greatly reduced.

[77] In an embodiment, the said method provides confident identification and confirmation of the peptide sequence and therefore the protein present in a sample. The selectivity and specificity are greatly enhanced due to the monitoring of all the transitions of a peptide in a single analysis. FIG. 1 illustrates the schematic representation of PRM (A) in a QqOrbitrap (HRMS) and pPRM (B) in a triple quad QqQ. In PRM, all product ions are monitored simultaneously, which correctly identifies and confirms the presence of the peptide from a complex biological sample, whereas in pPRM, product ions of the peptide, sufficient to confirm the sequence of the peptide is monitored. FIG 1).

[78] The tryptic peptides BT 1 and BT3 from the p chain of Hb were selected sincethe BT 1 peptide is the location of the Hb variant HbS, HbC and BT3 peptide is the location of HbE variant. BT12 from the p chain of Hb is the wild-type peptide for HbD and HbO variants. Doubly charged BT1 peptide (476.8m/z), BT3 peptide (657.8m/z), BT12 peptide (689.8m/z), HbS peptide (461.8m/z), HbC peptide (347.7m/z), HbE peptide (458.7m/z), HbD peptide (689.3m/z) and HbO peptide (625.3m/z) was selected in the first quadrupole and fragmented in the collision cell. [79] Table 1 illustrates BT1 peptide fragmentation table.

[80] Table 2 illustrates BT3 peptide fragmentation table.

[81] Table 3 illustrates BT12 peptide fragmentation table.

[82] Table 4 illustrates HbS peptide fragmentation table.

[83] Table 5 illustrates HbC peptide fragmentation table.

[84] Table 6 illustrates HbE peptide fragmentation table.

[85] Table 7 illustrates HbD peptide fragmentation table.

[86] Table 8 illustrates HbO peptide fragmentation table.

[87] Table 9 illustrates DT2 Lepore peptide fragmentation table.

[88] Table 10 illustrates Modified glycated Hb peptide fragmentation table.

B. EXAMPLE EMBODIMENTS:

[89] The present invention is illustrated in further detail by the following non-limiting examples.

[90] EXAMPLE 1: SCREENING OF HUMAN HEMOGLOBIN

[91] Inventors have used human hemoglobin as a model protein to demonstrate the invention. The mass of the protein is relatively small, less than 20kDa, which can be analyzed on a TQMS (Y. A. Daniel et al., 2005a). This protein ispresent in high amounts in the blood (g/dl blood). Several clinically important mutations of the protein exist among the human population (Huisman T, 1998), (Hb Var: A Database of Human Hemoglobin Variants and Thalassemias, n.d.) and the invention would help in confirming the presence or absence of these mutants of Hb in human blood.

[92] Commercially available Hemoglobin standard HbAo, comprising of a and β chains of Hb and HbS (sickle cell variant of Hb) was used to demonstrate the method. The protein standards HbAo and HbS were digested with trypsin according to the protocol (B. J. Wild, B.N.Green, E.K. Cooper, M.R.A.Lalloz, S.Erten, A.D. Stephens, 2001), resulting in the generation of hundreds of peptides. The enzyme specifically cleaves at the C terminal of Arginine and Lysine. The standards were tryptically digested and the tryptic peptide of wild type (BT1) and the variant HbS (STI) was selected in the first quadrupole and fragmented in the collision cell. The product ions are selected in the third quadrupole and detected by ESITQMS. [93] The many peptides thus generated were separated on a reverse phase UPLC column C18. The tryptic peptides BT1 and BT3 from the chain of HbAo standard were selected since the peptide is the location of the Hb variant HbS and HbE respectively. BT12 from the chain of HbAo standard is the wild-type peptide for HbD and HbO variants. Doubly charged BT1 peptide, 476.8m/z, BT3 peptide 657.8m/z, BT12 peptide, 689.8m/z and STI peptide 461.8m/z was selected in the first quadrupole and fragmented in the collision cell.

[94] A pseudoPRM method was created/set up to monitor all the transitions of the peptide with minimal optimization of collision energies and cone voltages. Collision-induced dissociation (CID) of peptides in the collision cell yields y ions (peptide fragments with charge retention on the C terminal region of the peptide) and b ions (peptide fragments with charge retention on the N terminalregion of the peptide).

[95] Analyzing the complete sequence of the peptide (y series fragments) confirms the presence of the protein. Monitoring b ion fragments adds specificity to the peptide sequence data. This method serves as an alternate technique to molecular gene sequencing for confirming the presence of the protein and its variants.

[96] The said pseudoPRM method was set up to obtain a complete sequence of the peptide for confident identification and confirmation of the Hb variants HbS, HbC, HbE, HbD, HbO, Hb Lepore and a modified glycated peptide of Hb. This is a simple and robust method for the qualitative analysis and confirmationof the presence of abnormal protein in a clinical sample.

[97] FIG. 2A-D illustrates the PseudoPRM of a tryptically digested Hb protein resulting in peptides BT1(A), BT3(B), BT12(C) (from β chain of HbAO standard) and ST1(D) peptide of HbS standard. FIG.2A illustrates Pseudo PRM spectra of tryptic peptide 1 of p chain of Hb [M+2H]2+, 476.8m/z from Hb standard HbAO. All the y ion fragment series (yl-y7) and b ion fragment series(b2-b4, b6) were observed confirming the presence of the chain of Hb. FIG.2B illustrates Pseudo PRM spectra of tryptic peptide 3 of chain of Hb [M+2H]2+,

657.8 m/z from Hb standard HbAO. All the y ion series fragments (yl-yl2) provide a complete sequence of the peptide. The b ion series fragments provide

additional confinnation of the peptide at the N terminal region confirming the presence of the p chain of Hb. FIG.2C illustrates Pseudo PRM spectra of tryptic peptide 12 of P chain of Hb [M+2H]2+, 689.8 m/z of Hb standard HbAO. All the y ion fragment series (yl-yll) and intense b2 and b3 ions confirm the presence of the P chain of Hb. FIG. 2D illustrates Pseudo PRM spectra of trypticpeptide STI of Hb [M+2H]2+, 461.8 m/z of HbS standard. Observation of the y ion series (yl-y7) indicates the complete sequence information of the variant peptide. The b ion series fragments provide additional confirmation of the peptide at the N terminal region thereby confirming the presence of HbS.

[98] The main advantage of the pseudoPRM method in a triple quadrupole Mass Spectrometer is to confirm the presence of a protein more confidently without the need for HRMS. If the clinical lab already possesses a triple quad instrument, the method can be easily implemented.

[99] The said method eliminates/postpones the need for high-resolution MS in a clinical or analytical laboratory for routine analysis. It also greatly reduces the cost of diagnosis for the patient/customer.

[0100] EXAMPLE 2: OPTIMISATION OF HUMAN HEMOGLOBINVARIANTS USING SYNTHETIC PEPTIDES

[0101] The proteotypic peptide (a peptide that is unique to the protein) that identifies Hb variants, HbS (VHLTPVEK), HbC (VHLTPK), HbE variant (VNVDEVGGK), HbD (QFTPPVQAAYQK) and HbO (FTPPVQAAYQ) were synthesized. The synthetic peptide was infused into the mass spectrometerwhile solvents were pumped through a separating device (Ultra high-pressure liquid chromatography) for optimizing the instrumental parameters like collision energy, cone voltage, and source parameters. A pseudoPRM method was developed based on the optimized parameters to monitor all the mass transitions of the peptide (y ion fragments) that depicts the complete sequence of the peptide. The resulting spectra are a direct readout of the sequence which confirms the presence of the defective protein (HbS, HbC, HbE, HbD, HbO). For HbD, the y ion series are not diagnostic (Haynes et al., 2013) since they are also present in the wild-type BT12 peptide. Since the mutation (Glu to Gin) is at the N terminal region, b2 and b3 ion were monitored, which confirms the presence of the variant protein HbD. For HbO, the mass of the peptide is smallerthan the BT12 peptide even though all yl-ylO ion series are the same as BT12.‘b’ ion series are different and hence its presence can be diagnostic of the variantHbO.

[0102] FIG. 3A-E illustrates the PseudoPRM spectra of the synthesized peptides representing Hb variants HbS(A), HbC(B), HbE(C), HbD(D) and HbO(E) according to the aspects of the invention. FIG. 3A illustrates Pseudo PRM spectra of synthetic HbS peptide of Hb [M+2H]2+, 461.8 m/z. Optimization of cone voltage and collision energies to yield all product ions of HbS peptide using synthetic HbS. FIG. 3B illustrates Pseudo PRM spectra of synthetic HbC peptide of Hb [M+2H]²⁺, 347.7 m/z. Optimization of cone voltage and collision energies to yield all product ions of HbC peptide using synthetic HbC. Since it is a small peptide of 6aminoacids, both ion types b and y were observed to improve confident identification. yl-y5 and b2-b4 ions are observed which provides information of the sequence of the peptide in both directions N-C terminal and C-N terminal. FIG. 3C illustrates Pseudo PRM spectra of synthetic HbE peptide of Hb [M+2H]²⁺, 458.7 m/z. Optimization of cone voltage and collision energies to yield all product ions of HbE peptide using synthetic HbE. FIG. 3D illustrates Pseudo PRM spectra of synthetic HbDpeptide of Hb [M+2H]²⁺, 689.4 m/z. Optimization of cone voltage and collision energies to yield all product ions of HbD peptide using synthetic HbD. FIG. 3Eillustrates Pseudo PRM spectra of synthetic HbO peptide of Hb [M+2H]²⁺,

625.3 m/z. Optimization of cone voltage and collision energies to yield all product ions of HbO peptide using synthetic HbO. y5-y7 ions have been magnified 5 times to reveal the ions.

[0103] EXAMPLE 3: EXPERIMENTAL METHODOLOGIES

[0104] According to an embodiment, Hemoglobin protein is digested with trypsin to generate peptides. These peptides are subjected to UPLC where the peptides get separated and are eluted from the column under gradient conditions. The eluted peptides are ionized in the electrospray source region of the TQMS. Alternatively, tryptic peptides can be introduced directly intoTQMS for ionisation of the peptides. The first quadrupole selects the proteotypic peptide (peptides unique to the protein or Hb variant) which is fragmented in the collision cell. The fragments/product ions are selected in thethird quadrupole and are detected by MS. The mass to charge ratio (m/z) of theproduct ions and the proteotypic peptide at a specific retention time (RT), confirms the presence of the protein.

[0105] EXAMPLE 4: EXAMPLE EMBODIMENTS OF THE PROTEINSEOUENCES

[0106] Uniprot protein sequence of β chain of Human Hemoglobin -

P68871 a. VHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESF

GDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDK

LHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH

[0107] Sequence of Hemoglobin S variant - β chain of HumanHemoglobin -

[0108] Natural variant VAR_002863 b. VHLTPVEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESF

GDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDK

LHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH.

[0109] Sequence of Hemoglobin C variant - chain of HumanHemoglobin -

[0110] Natural variant VAR_002864

[0111] VHLTPKEKS A VT ALWGKVN VDE VGGE ALGRLLV V YPWTQR

FFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFAT

LSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVV AGVANALAHKYH

[0112] Sequence of Hemoglobin E variant - chain of HumanHemoglobin -

[0113] Natural variant VAR_002907 c. VHLTPEEKSAVTALWGKVNVDEVGGKALGRLLVVYPWTQRFFESF

GDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDK

LHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH

[0114] Sequence of Hemoglobin D variant - P chain of HumanHemoglobin -

[0115] Natural variant VAR_003048 d. VHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESF GDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDK LHVDPENFRLLGNVLVCVLAHHFGKQFTPPVQAAYQKVVAGVANALAHKYH

[0116] Sequence of Hemoglobin O variant - chain of HumanHemoglobin - [0117] Natural variant VAR_003049. Site of mutation is highlighted. e. VHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESF GDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDK LHVDPENFRLLGNVLVCVLAHHFGKKFTPPVQAAYQKVVAGVANALAHKYH

[0118] C, IMPORTANT ATTRIBUTES OF THE INVENTION

[0119] A simple and robust method is developed which provides a complete sequence of the peptide in a TQMS-low resolution mass spectrometer. The instrumental parameter optimization is done in an automated fashion, which does not require elaborate tuning for qualitative analysis of the peptides. All mass transitions comprising the complete sequence of the peptide can bevisualized in a single spectrum. This brings in additional specificity than the 3-5 transitions that are monitored for quantitative assays on a TQMS.

[0120] The spectra are a direct read-out of the data and no additional software is required to analyze the data. There is no requirement for an expert to interpret the data. The complete sequencing of the proteotypic peptide of HbS, HbC, HbE, HbD and HbO variant of p chain of hemoglobin, in a TQMS, is demonstrated with synthetic peptide (FIG 3A-E) and validated using patient samples (Fig 4A-D). This invention avoids the need for HRMS or gene

sequencing for confirming the presence of the defective protein in a patient’s sample.

[0121] The current invention provides complete sequence information of the peptide for qualitative analysis of proteins with improved selectivity.

[0122] USES, APPLICATIONS AND BENEFITS OF THEINVENTION

[0123] Invention provides following benefits,

[0124] Analysis of Hemoglobin variants:

[0125] CONFIRMATION of the PRESENCE of Hb variants identified during the screening of hemoglobinopathies.

[0126] Screening of Hemoglobinopathies is performed Using HPLC/UPLC and/or Capillary electrophoresis (CE). Sample processing is performed manually. Figs 4A-D illustrate confirmation of the presence of Hb variants using the invention. The sample type is whole blood.

[0127] DBS is the main sample type for diagnosing various inborn errors ofmetabolism for newborns (Neonates). Screening for hemoglobinopathies is an extended application (newborn screening NBS), currently performed using HPLC / LCMS / CE with manual processing or a sample preparation kit (spot-on diagnostic kit). Samples that were positive during screening, can be CONFIRMED for the PRESENCE of the variant using the pseudoPRM method using ESI TQMS.

[0128] CONFIRMATION of the PRESENCE of Hb variants from different sample types - dried blood spots (DBS) from new born babies has been demonstrated. Figs 5 illustrate confirmation of the presence of Hb variants (HbS, HbC, HbE, HbD) using the invention. The sample type is DBS from newborn babies.

[0129] Apart from clinically significant Hb variants (S, C, E, D, O) any otherHb variants of concern can also be confirmed (e g., Lepore, HbJ, HbQ, a, 5 chain variants etc.). Fig 6 illustrates the confirmation of presence of Hb Leporein a patient's sample. Fig 7 illustrates the confirmation of a glycated peptide ofHb.

[0130] Insulin

[0131] Patients requiring daily insulin treatment require monitoring of their insulin levels to control or manage diabetes. There are several analogs of recombinant insulin available on the market. So, it is important to know the quantity of insulin taken as a drug that is different from human insulin producedby the pancreas. [0132] Insulin administration has a forensic interest too, in wrongful death cases.

[0133] Insulin can also be used as a performance-enhancing drug and is measured in doping tests

[0134] The current invention, the pseudoPRM method using ESI TQMS, can be utilized to CONFIRM THE PRESENCE of the various analogs of insulin with or without the digestion of insulin.

[0135] Analysis of Immunoglobulins IgGs-M proteins

[0136] Assessing and monitoring M proteins or spike proteins helps in the diagnosis of plasma cell disorders.

[0137] M proteins are extracted from serum by using specific antibodies. Upon digestion of the extracted M proteins, proteospecific (peptide specific to the isotype of immunoglobulins) peptides are selected and fragmented in the collision cell of TQMS.

[0138] Monitoring the product ions provides CONFIRMATION of the PRESENCE of the isotype of Igs

[0139] Qualitative analysis of MTb peptides:

[0140] The detection and management of MTb infections are fraught with challenges partly due to the currently available microbiologic techniques which have moderate sensitivity, specificity and a long turnaround time.

[0141] The present invention can detect the PRESENCE of active MTb infections by monitoring the MTb- specific peptides of lOkDa culture filtrate protein (CFP-10) and 6kDa early secretory antigenic target (ESAT-6) more confidently with increased sensitivity and selectivity.

[0142] Performance enhancing Bioactive Peptides - Doping tests

[0143] Bioactive peptides are administered to enhance the performance of sportspersons and to horses for recreation purposes (horse racing).

[0144] Mass spectrometry plays a major role in detecting these peptides.

[0145] The current invention can CONFIRM the PRESENCE of theperformance- enhancing peptides. [0146] Egs include growth hormone -releasing peptides and their analogs, ipamorelin, CJC-1295 and rHGH.

[0147] In additional embodiments, the presented invention can be applied to,

[0148] Multiplexing of other peptides/proteins in the same method is possible.

[0149] The invention can be widely used in all low-resolution MS instruments, from other vendors.

[0150] The pseudoPRM method can be extended to identify other proteins of significance in clinical or analytical laboratories.

[0151] If the quantitation of the protein is not compromised, the same method can be used to measure the protein qualitatively and quantitatively - a potential application in targeted proteomics.

[0152] The method can be used by academicians, where researchers require only qualitative analysis of various proteins with improved selectivity.

[0153] The method provides a quick way of checking the quality of the recombinant proteins either from a commercial source or from manufacturers of different therapeutic proteins.

[0154] Other industries like biotech, pharma, toxicology etc... also benefit from this method if used in a quality control environment.

[0155] BEST MODE TO PRACTICE

[0156] The invention can be translated to clinical laboratories for population screening of clinically important Hb variants with more selectivity. The current method can replace the molecular confirmation of clinically relevant Hb variants like HbS, HbC, HbE, HbD, HbO, at a more affordable cost to the patient. If the sequence of other rare variants is known, it can be confirmed as well.

[0157] The method can be used by blood banks to check for Hb variants in the donor’s blood.

[0158] The method can also be used to confirm the presence or absence of analogous proteins, which are different than the endogenous protein if their masses are different (eg, endogenous insulin vs insulin drugs, endogenous monoclonal antibodies vs therapeutic antibodies). [0159] The method will be useful in the biotech and pharma industry as a quality control measure to check the quality of the peptide/protein drugs duringthe manufacturing process.

[0160] Merely for illustration, only representative number/type of graph, chart, block, and sub-block diagrams were shown. Many environments often contain many more block and sub-block diagrams or systems and sub-systems, both in number and type, depending on the purpose for which the environment is designed.

[0161] While specific embodiments of the invention have been shown and described in detail to illustrate the inventive principles, it will be understood that the invention may be embodied otherwise without departing from such principles.

[0162] Reference throughout this specification to “one embodiment”, “an embodiment”, or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment”, “in an embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0163] It should be understood that the figures and/or screenshots illustrated in the attachments highlighting the functionality and advantages of the present invention are presented for example purposes only. The present invention is sufficiently flexible and configurable, such that it may be utilized in ways other than that shown in the accompanying figures.

[0164] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are tobe included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

29 We claim.

1. A method for screening I analyzing / confirming a protein variant in a sample comprising:

(i) processing a protein to produce a series of peptides;

(ii) ionizing the series of peptides in a source region of Electrospray ionization Tandem Triple Quad Mass Spectrometer;

(iii) selecting proteotypic peptide ion specific to the protein variant in a first quadrupole and fragmenting the selected proteotypic peptide ion in a collision cell;

(iv) selecting greater than five product ions representative of the proteotypic peptide ion in pseudo-Parallel Reaction Monitoring mode by a third quadrupole;

(v) monitoring the selected product ions by a detector; and

(vi) detecting the presence of the peptide using the Electrospray ionization Tandem Triple Quad Mass Spectrometer, wherein a mass/charge ratio (m/z) of the product ions and the proteotypic peptide ion indicates the presence of the protein variant in the sample.

2. The method of claim 1, wherein the method includes visualizing the mass spectra obtained by the Electrospray ionization Tandem Triple Quad Mass Spectrometer, wherein the mass spectra are a direct readout of the sequence of the peptide.

3. The method of claim 1, wherein the quadrupole scans all proteotypic peptide ions at unit resolution with a scan time of 0.1-0.005 seconds.

4. The method of claim 1, wherein the protein variant is a hemoglobin variant, insulin, Immunoglobulins, MTb peptides, and membrane proteins.

5. The method of claim 4, wherein the hemoglobin variant is HbS, HbC, HbE, HbD, HbO, Hb Lepore, glycated Hb.

6. The method of claim 1, wherein the protein is processed by digestion, wherein the protein is digested with N-tosyl-L-phenylalanine chloromethyl ketone treated trypsin. 30

7. The method of claim 1, wherein the sample is a biological sample, wherein the biological sample is Dried Blood spot (DBS), whole blood spotted onto Dried blood spot (DBS), urinary protein, Cerebro spinal fluids, and body fluids.

8. The method of claim 1, wherein the sample is a protein or peptide extracted from tissues, body fluids, cells of an animal, plant, fungus, bacteria or other microorganisms, wherein the sample is a chemically synthesized peptide.

9. A method for screening I analyzing I confirming a protein variant in a sample comprising:

(i) processing a protein to produce a series of peptides;

(ii) subjecting the series of peptides into a liquid chromatography reverse phase column;

(iii) separating the peptides in the liquid chromatography reverse phase column under gradient conditions;

(iv) ionizing the separated peptides in a source region of Electrospray ionization Tandem Triple Quad Mass Spectrometer;

(v) selecting proteotypic peptide ion specific to the protein variant in a first quadrupole and fragmenting the selected proteotypic peptide ion in a collision cell;

(vi) selecting greater than five product ions representative of the proteotypic peptide ion in pseudo-Parallel Reaction Monitoring mode by a third quadrupole;

(vii) monitoring the selected product ions by a detector; and

(viii) detecting the presence of the peptide using the Electrospray ionization Tandem Triple Quad Mass Spectrometer, wherein a mass/charge ratio (m/z) of the selected product ions and the proteotypic peptide ion eluting at a specific retention time (RT) indicates the presence of the protein variant in the sample.

10. The method of claim 9, wherein the method includes visualizing the mass spectra obtained by the Electrospray ionization Tandem Triple Quad Mass Spectrometer, wherein the mass spectra are a direct readout of the sequence of the peptide.

11. The method of claim 9, wherein the method includes using synthetic peptides to optimize peptide fragmentation patterns.

12. The method of claim 11, wherein the synthetic peptides are diluted in Liquid Chromatography-Mass Spectroscopy solvent without tryptic digestion to improve ionization of the peptides.

13. The method of claim 12, wherein the Liquid Chromatography -Mass Spectroscopy solvent is acidified acetonitrile.

14. The method of claim 9, wherein the gradient conditions are between 20-40% Acetonitrile, <0.5% Formic acid in 1-3.5 mins.

15. The method of claim 9, wherein the specific retention time (RT) comprises between 1.0- 3.5 minutes depending on the peptide variant.

16. The method of claim 9, wherein the liquid chromatography reverse phase column is an ultra-pressure liquid chromatography reverse phase column.

17. The method of claim 9, wherein the quadrupole scans all proteotypic peptide ions at unit resolution with a scan time of 0.1-0.005 seconds.

18. The method of claim 9, wherein the protein variant is a hemoglobin variant, insulin, Immunoglobulins, MTb peptides, and membrane proteins.

19. The method of claim 18, wherein the hemoglobin variant is HbS, HbC, HbE, HbD, HbO, Hb Lepore, glycated Hb.

20. The method of claim 9, wherein the protein is processed by digestion, wherein the protein is digested with N-tosyl-L-phenylalanine chloromethyl ketone treated trypsin and diluted in the Liquid Chromatography -Mass Spectrometer.

21. The method of claim 9, wherein the sample is a biological sample, wherein the biological sample is whole blood, Dried Blood spot (DBS), whole blood spotted onto Dried blood spot (DBS), urinary protein, Cerebro spinal fluids, and body fluids.

22. The method of claim 9, wherein the sample is a protein or peptide extracted from tissues, body fluids, cells of an animal, plant, fungus, bacteria or other microorganisms, wherein the sample is a chemically synthesized peptide.