Advancements in clinical approaches, analytical methods, and smart sampling for LC-MS-based protein determination from dried matrix spots.

Determination of proteins from dried matrix spots using MS is an expanding research area. Mainly, the collected dried matrix sample is whole blood from a finger or heal prick, resulting in dried blood spots. However as other matrices such as plasma, serum, urine, and tear fluid also can be collected in this way, the term dried matrix spot is used as an overarching term. In this review, the focus is on advancements in the field made from 2017 up to 2023. In the first part reviews concerning the subject are discussed. After this, advancements made for clinical purposes are highlighted. Both targeted protein analyses, with and without the use of affinity extractions, as well as untargeted, global proteomic approaches are discussed. In the last part, both methodological advancements are being reviewed as well as the possibility to integrate sample preparation steps during the sample handling. The focus, of this so-called smart sampling, is on the incorporation of cell separation, proteolysis, and antibody-based affinity capture.

Smart sampling as the "Spot-on" Method for LC-MS protein analysis from dried blood spots.

This perspective explores the feasibility of smart sampling with dried blood spots for the determination of proteins and peptides from human biomatrices using liquid chromatography coupled to mass spectrometry for clinical purposes. The focus is on innovative approaches to transform filter paper from a mere sample carrier to an active element in sample preparation, with the aim of reducing the need for extensive and intensive sample preparation in the conventional sense. Specifically, we discuss the use of modified cellulose to integrate sample preparation at an early stage of sample handling. The use of paper immobilized with either trypsin or monoclonal antibodies for protein digestion and affinity clean-up is discussed as a potential benefit of starting sample preparation instantly at the moment of sampling to optimize time efficiency and enable faster analysis, diagnosis, and follow-up of patients.

The utility of molecularly imprinted polymers for mass spectrometric protein and proteomics analysis.

Selective and efficient sample clean-up is important in mass spectrometric protein- and proteomics analyses from biological matrices. Molecularly imprinted polymers (MIPs), polymers prepared to have tailor-made cavities for capture of target analytes may by such represent an interesting alternative for selective clean-up. The present review aims to give an overview of the utility of MIPs for protein capture from biological matrices prior to mass spectrometry (MS) analysis. The application of MIPs in depletion of abundant proteins, in protein and proteotypic peptide capture as well as in capture of post-translational modifications (PTMs) is described and discussed. In addition, an overview of available MIP formats and their advantages and challenges is given, together with an overview of the mass spectrometric techniques used in protein analysis after MIP capture. Overall, the present literature demonstrates that for many applications MIPs for sample clean-up in mass spectrometric protein and proteomics analysis from biological matrices is still not fully matured. MIPs for proteotypic peptide capture is the most mature approach and a method for routine use may be available within the next few years.

On the spot immunocapture in targeted biomarker analysis using paper-bound streptavidin as anchor for biotinylated antibodies.

The modification of an easily available resource like paper to circumvent expensive or intensive sample pretreatment could be the answer to sample analysis in resource-poor regions. Therefore, a novel on-paper device combining sample collection with affinity sample pretreatment is introduced here. Universal smart affinity samplers are produced by a simple KIO4-mediated oxidation of cellulose, which functionalizes the paper. This is followed by immobilization of streptavidin. Streptavidin serves as a universal anchor for biotinylated antibodies, enabling simple preparation of tailor-made affinity samplers. The functionality of the device was tested using a model protein (human chorionic gonadotropin, hCG) and biotinylated anti-hCG antibodies for affinity capture. In a laboratory setting, the performance was demonstrated, and a 14-fold increase of target binding compared to binding without bmAb was achieved. The recovery of hCG captured with bmAb-treated samplers was determined to be 33% and comparable to previously described affinity capture approaches. Application of the smart affinity samplers to human serum containing hCG showed an R2 of 0.98 (200-1000 pg mL-1), precision of ≤ 9.1% RSD, and estimated limit of detection of 65 pg mL-1. Although further optimization and validation are necessary prior to application to real samples in clinical settings, the potential of the device for use in determination of low abundant biomarkers in complex samples has been demonstrated.

Matrix-assisted ionization mass spectrometry in targeted protein analysis - An initial evaluation.

RATIONALE: Matrix-assisted ionization (MAI) is a relatively new ionization technique for analysis by mass spectrometry (MS). The technique is simple and has been shown to be less influenced by matrix effects than e.g. electrospray ionization (ESI). These features are of interest in the targeted analysis of proteins from biological samples. METHODS: Targeted protein determination by MAI-MS was evaluated using a triple quadrupole mass analyzer equipped with a stripped nanoESI source in selected reaction monitoring (SRM) mode. The proteins were analyzed using the bottom-up approach with stable isotopic labeled peptides as internal standards (IS). The MAI matrix was 3-nitrobenzonitrile dissolved in acetonitrile. Aqueous sample and matrix solution were mixed in a 1:3 volume ratio. One microlitre of the dried matrix/analyte sample was introduced into the inlet of the mass spectrometer where ionization commences. RESULTS: SRM settings established for ESI-SRM-MS of the peptides here investigated were applicable in MAI-SRM-MS for all evaluated peptides except one that is poorly soluble in water. Addition of IS provided efficient correction at most levels (relative standard deviation (RSD) ≤28% (except lowest digest level), r2 ≥ 0.995). This was also true for the more complex biological matrices, diluted urine (1:1; RSD = 20% a synthetic peptide, NLLGLIEAK) and diluted digested serum (1:100; RSD = 7% digested cytochrome C). Biological matrix influenced the signal intensity unless sufficiently diluted. CONCLUSIONS: The results demonstrate that MAI-SRM-MS has promising potential in targeted protein determination by the bottom-up approach because of its simplicity, ease of use, and speed. However, more data is needed to confirm the results prior to application in a clinical setting.

Magnetic Synthetic Receptors for Selective Clean-Up in Protein Biomarker Quantification.

Biomarker analysis by mass spectrometry (MS) can allow for the rapid quantification of low abundant biomarkers. However, the complexity of human serum is a limiting factor in MS-based bioanalysis; therefore, novel biomarker enrichment strategies are of interest, particularly if the enrichment strategies are of low cost and are easy to use. One such strategy involves the use of molecularly imprinted polymers (MIPs) as synthetic receptors for biomarker enrichment. In the present study, a magnetic solid-phase extraction (mSPE) platform, based on magnetic MIP (mMIP) sorbents, is disclosed, for use in the MS-based quantification of proteins by the bottom-up approach. Progastrin releasing peptide (ProGRP), a low abundant and clinically sensitive biomarker for small cell lung cancer (SCLC), was used to exemplify the mSPE platform. Four different mMIPs were synthesized, and an mSPE method was developed and optimized for the extraction of low concentrations of tryptic peptides from human serum. The mSPE method enabled the selective extraction of the ProGRP signature peptide, the nonapeptide NLLGLIEAK, prior to quantification of the target via LC-MS/MS. Overall, the mSPE method demonstrated its potential as a low cost, rapid, and straightforward sample preparation method, with demonstrably strong binding, acceptable recoveries, and good compatibility with MS. mMIPs are a potential low-cost alternative to current clinical methods for biomarker analysis.

Smart blood spots for whole blood protein analysis.

A reactor for whole blood sampling integrated with instant protein digestion in a "lab-on-paper" format is introduced here. The sampling reactor was fabricated on commercially available filter paper using 2-hydroxyethyl methacrylate-co-2-vinyl-4,4-dimethyl azlactone (HEMA-VDM) polymerization followed by the immobilization of trypsin. Immobilization conditions were investigated with respect to temperature, enzyme amount and immobilization time. The highest reactor efficiency with respect to protein digestion was obtained with 1.25 mg trypsin per reactor immobilized at room temperature for 3 hours. Commercially available cellulose filter papers and DMPK-C cards were modified and immobilized with trypsin prior to whole blood sampling. Filter paper specifications including thickness (180-220 µm), weight (77-92 g m-2) and porosity (11-25 µm) were investigated with respect to performance (digestion efficiency and extraction recovery). From this study, it was found that a medium thickness paper with higher weight and porosity is optimal for reactor efficiency. The reactors were tested and compared with respect to a standard dried blood spot procedure for protein digestion. The most efficient reactors obtained 134 ± 14 and 124 ± 7 high confidence protein groups for freeze thawed and fresh whole blood samples, respectively.

Correction to: Exploring the peptide retention mechanism in molecularly imprinted polymers.

The authors would like to call the reader's attention to the fact that unfortunately due to the file formatting during the exporting of the data matrix from the program Unscrambler (used for the development of the statistical model) to the Word office file.

Instant on-paper protein digestion during blood spot sampling.

A concept integrating sampling and protein digestion is introduced here combining fast and simple fabrication by wax printing on filter paper with trypsin immobilized polymer beads. The paper reactors showed promising results with a high degree of protein digestion within fifty minutes in model protein mixtures as well as in human blood. The model protein mixture was used for the evaluation of performance both with and without a reduction and alkylation step. The paper reactors without reduction and alkylation showed between 46% and 75% protein sequence coverage and between five and 20 high confidence peptides (one and five zero missed cleavage peptides, respectively). Compared to a conventional in-solution approach, the paper reactor showed 10% less protein sequence coverage, 29% fewer high confidence peptides and 19% fewer high confidence peptides with zero missed cleavages. Placement of the protein reduction and alkylation step (before or after protein digestion) was shown to be of low importance. The storage stability of the paper reactors with (six weeks) and without (twelve weeks) tryptic peptides was satisfactory. The ability of the paper reactors to digest complex biological samples was investigated by comparison with human whole blood samples prepared using a conventional dried blood spot (DBS) procedure with overnight digestion in non-targeted analysis. The reactors showed a comparable performance with 75 ± 25 for the protein groups compared to 76 ± 5 for the DBS samples. Additionally, 267 ± 72 and 335 ± 11 unique peptides (high confidence) were identified for on-paper digestion and DBS, respectively.

Expanding the knowledge on dried blood spots and LC-MS-based protein analysis: two different sampling materials and six protein targets.

The combination of dried blood spots (DBS) and bottom-up LC-MS-based protein analysis was investigated in the present paper using six model proteins (1 mg/mL of each protein) with different physicochemical properties. Two different materials for DBS were examined: a water-soluble DBS material (carboxymethyl cellulose, (CMC)) and a commercially available (non-soluble) material (DMPK-C). The sample preparation was optimised regarding the water-soluble material and achieving acceptable repeatability of the signal was emphasised. Five microlitres of whole blood were deposited and dried on either CMC or DMPK-C. The samples were dissolved (CMC) or extracted (DMPK-C) prior to tryptic digest and matrix precipitation. The optimization of the sample preparation showed that an increased buffer concentration (100 mM ammonium bicarbonate) for dissolving the DBS samples gave better repeatability combined with a decrease in analyte signal. CMC seemed to add extra variability (RSD 8-60%) into the analysis compared to sample prepared without CMC (RSD 6-36%), although equal performance compared to DMPK-C material (RSD 13-60%) was demonstrated. The stability of the analytes was examined for different storage periods (1 and 4 weeks) and different storage temperatures (-25, 25, and 40 °C). The stability on both CMC (> 70% compared to reference) and DMPK-C (> 50% compared to reference) was acceptable for most of the peptides. This paper shows that both DBS materials can be used in targeted LC-MS-based protein analysis of proteins with different physicochemical properties. Graphical Abstract Overview of the experimental set-up for expanding the knowledge of dried blood spots in LC-MS-based protein anaysis.

Exploring the peptide retention mechanism in molecularly imprinted polymers.

Molecularly imprinted polymers (MIPs) have been used as useful sorbents in solid-phase extraction for a wide range of molecules and sample matrices. Their unique selectivity can be fine-tuned in the imprinting process and is crucial for the extraction of macromolecules from complex matrices such as serum. A relevant example of this is the application of MIPs to peptides in diagnostic assays. In this article the selectivity of MIPs, previously implemented in a quantitative mass-spectrometric assay for the biomarker pro-gastrin-releasing peptide, is investigated. Partial least squares regression was used to generate models for the evaluation and prediction of the retention mechanism of MIPs. A hypothesis on interactions of MIPs with the target peptide was verified by ad hoc experiments considering the relevant peptide physicochemical properties highlighted from the multivariate analysis. Novel insights into and knowledge of the driving forces responsible for the MIP selectivity have been obtained and can be directly used for further optimization of MIP imprinting strategies. Graphical Abstract Applied analytical strategy: the Solid Phase Extraction (SPE) of digested Bovin Serum Albumin (BSA), using Molecularly Imprinted Polymers (MIP), is followed by the liquid chromatography-mass spectrometry (LC-MS) analysis for the identification of the retained peptides. The further application of multivariate analysis allows setting up a Partial Least Square (PLS) model, which describes the peptide retention into the MIP and gives additional knowledge to be used in the optimization of the MIP and the whole SPE method.

Dual-immuno-MS technique for improved differentiation power in heterodimeric protein biomarker analysis: determination and differentiation of human chorionic gonadotropin variants in serum.

If the biomarker potential of intact heteromers and their free subunits is different, differentiation between these forms may reveal important clinical information. Such differentiation may however be analytically challenging. One possible way of circumventing this challenge is by performing a dual-immuno-MS approach. In the present paper, a two-step immunoaffinity sample preparation step is succeeded by digestion and subsequent LC-MS analysis to provide high-sensitivity quantification and differentiation between the heterodimer human chorionic gonadotropin (hCG) and its free ß-subunit in serum. Intact and free variants are captured in two separate immunoextraction steps in order to increase the differentiation power of the method. Intact heterodimer variants were depleted prior to free subunit variants in order to incorporate a method quality control. The method was optimized for serum samples. A fully validated immuno-MS method was used as foundation, and partial validation according to the European Medicines Agency's (EMA) guidelines on validation of bioanalytical methods was performed for the dual approach. An accelerated digestion step was incorporated making batch processing of samples within 1 day possible (approx. 3.5 h of sample preparation including digestion). Acceptable linearity (R (2) ≥ 0.990 for four variants and R (2) of 0.920 and 0.966 for the remaining two) and specificity were demonstrated, and the method was robust toward varying levels of intact heterodimer versus free subunit. The method was also successfully tested on realistic samples, demonstrating both the differences in total hCG and the distribution between intact hCG and its free ß-subunit in real samples. Graphical abstract Schematic overview of the dual immuno-MS process.

Water-Soluble Dried Blood Spot in Protein Analysis: A Proof-of-Concept Study.

In the present work human chorionic gonadotropin (hCG) was used as a model protein in a proof-of-concept study combining water-soluble dried blood spot (DBS) material in liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based protein analysis. A water-soluble material consisting of commercially available carboxymethyl cellulose (CMC) was evaluated as sampling material for this purpose. The material dissolved readily at physiological pH. Different sample preparation methods were evaluated, and in the final method, 15 µL of whole blood was deposited and dried on CMC before the whole spot was dissolved prior to cleanup by immunoaffinity extraction, tryptic digest, and preconcentration by solid-phase extraction (SPE). The results indicated complete dissolution of hCG from the spots, acceptable limit of detection (LOD) (0.1 IU/mL), linearity (R(2) = 0.959), accuracy (16%), and precision (≤22%). Long-term stability (45 days) of hCG in dried spots at reduced temperatures (≤8 °C) was also demonstrated. The analyte recovery was comparable to the commercially available nonsolvable cellulose material (FTA DMPK-C card).

Why less is more when generating tryptic peptides in bottom-up proteomics.

Proteolytic digestion is a time consuming and critical step in bottom-up proteomic analysis. The most widely used protease, trypsin, has high specificity and generates peptides that are considered to be ideally suited for bottom-up LC-MS technology. By exploiting key factors affecting enzymatic activity we obtained a simple, straightforward, and rapid in-solution digest protocol that performed better than the conventional overnight digestion method in terms of amino acid coverage of proteins, number of peptides generated, and peptide ion abundances. Prolonged digestion time, such as overnight digestion, leads to decline in protein amino acid coverage and loss of tryptic peptides. This was found to be caused by complete digestion by trypsin leading to an increased number of small peptides that are not LC-MS detectable. Slow-rate nontryptic digestion of peptides is a contributing factor for loss of peptide ion intensities during extended digestion time. Our work demonstrates that for both qualitative and quantitative bottom-up proteomic studies it is beneficial to prevent trypsin digestion to go to completion by reducing treatment time from the conventional several hours to a few minutes cleavage time.

Antibody-free biomarker determination: exploring molecularly imprinted polymers for pro-gastrin releasing peptide.

Biomarker mass spectrometry assays are in high demand, and analysis of pro-gastrin releasing peptide (ProGRP) as a small cell lung cancer marker has been recently investigated by mass spectrometry after immunoextraction. In this article, we introduce an assay based on molecularly imprinted polymers (MIPs) targeting the proteotypic peptide of ProGRP as a possible alternative to current immuno-based assay. The MIPs were prepared by surface-initiated reversible addition-fragmentation chain transfer polymerization and were introduced as sorbents for the cleanup and enrichment of a ProGRP signature peptide from tryptically treated serum samples. The use of an appropriate solid-phase extraction protocol allowed specific extraction of the target peptide while depleting other peptides that arose from the sample digestion, hence resulting in reduced background. The selective extraction of a ProGRP signature peptide, after digestion of serum samples, translates into a time- and cost-effective method suited for bottom-up analysis wherever targeted peptide extraction from complex matrices is required.

Epitope analysis and detection of human chorionic gonadotropin (hCG) variants by monoclonal antibodies and mass spectrometry.

Human chorionic gonadotropin (hCG) is an important marker for pregnancy, pregnancy-related disorders, and various cancers. Different molecular forms of hCG occur in different clinical conditions, and these can be distinguished with immunoassays using well-characterized monoclonal antibodies. Exact knowledge of the epitopes of the antibodies used is crucial for the design of assays with desired specificity. The epitopes of many hCG antibodies have been determined by comparing their reactivity with six 1st International Reference Reagents (IRRs) for hCG, but the specificity of some antibodies remains to be exactly defined. We have therefore studied the reactivity of 30 monoclonal antibodies (mAbs) with the six 1st IRRs for hCG, and variants were investigated using immunoaffinity extraction combined with liquid chromatography-mass spectrometry (LC-MS/MS) for the detection of hCG variants by specific tryptic signature peptides. Each of the mAbs had previously been characterized with regard to epitope specificity in the 2nd Tissue Differentiation Workshop on hCG of the International Society of Oncology and BioMarkers (ISOBM). Simultaneous identification of different hCG variants by LC-MS/MS confirmed that two standards used for mAb characterization, nicked hCG (hCGn, 1st IRR 99/642) and nicked ß subunit of hCG (hCGßn, 1st IRR 99/692), are heterogeneous, being composed of two major variants each: hCGn44/45 and hCGn47/48 as well as hCGßn44/45 and hCGß47/48. Furthermore, MS revealed cross-contamination by non-nicked hCG of the 1st IRR hCGn (99/642) standard. This information enabled fine-tuning of the previous epitope classifications of mAbs specific for heterodimeric hCG (c-mAbs). LC-MS/MS confirmed that c2-mAbs and most c1-mAbs did not recognize hCGn as the observed response in radioimmunoassays obviously resulted from the contamination of hCGn with hCG. Thus, c1 and c2 epitopes are partially dependent on hCGß peptide loop 2. c3-mAbs recognized both hCG and hCGn. It appeared that c-mAbs cannot discriminate between hCGn44/45 and hCGn47/48 as they either recognize both or neither variant. For most mAbs directed against hCGß, epitope specificity determined by LC-MS/MS was highly concordant with that obtained using standard immunological methods. In analogy to c-mAbs, hCGß-mAbs cannot discern between hCGßn44/45, hCGßn47/48, or intact hCGß as all 15 mAbs recognizing hCGß also recognized both nicked variants irrespective of which of the three major hCGß antigenic domains their epitopes were located within: on the caps of peptide loops 1 and 3, around the cystine knot, or along the hCGßCTP. LC-MS/MS confirmed that their epitopes were not located on hCGß peptide loop 2. Thus, LC-MS/MS provided in-depth information on hCG variant composition of hCGn (99/642) and hCGßn (99/692) and hCG variant specificity profiles and facilitated precise classification of the epitopes of anti-hCG mAbs. This has impact on the design of selective immunoassays.

Determining ProGRP and isoforms in lung and thyroid cancer patient samples: comparing an MS method with a routine clinical immunoassay.

This paper compares two methods to determine the tumor marker progastrin-releasing peptide (ProGRP): as routine assay, the automated time-resolved immunofluorometric assay (TR-IFMA), which allows total ProGRP determination; and the immunocapture liquid chromatography selected reaction monitoring mass spectrometry (LC-SRM-MS) method, which additionally allows isoform differentiation. The investigation included 60 serum samples from patients suffering from various cancer diseases which may cause elevated ProGRP levels (small cell lung carcinoma; SCLC, non-small cell lung carcinoma; NCLC; and medullary thyroid cancer; MTC, as well as some with unspecific diagnosis). The two methods showed good correlation (R (2) = 0.887). However, the MS method determines the total ProGRP concentration systematically approximately 30 % lower than the TR-IFMA, implying that the absolute values generated by the methods are not interchangeable. The MS method gives additional information about isoform levels in the samples, providing novel insight into isoform expression on the protein level.

One-step functionalization of paper and simplified antibody immobilization for on-the-spot immunocapture from dried serum in liquid chromatography-tandem mass spectrometry based targeted protein determination.

This work aimed to simplify and improve the process of binding monoclonal antibodies (mAbs) covalently to filter paper for use in dried blood spot sampling, enabling instant capture of protein biomarkers for targeted protein determination. Incorporating the necessary immunocapture sample preparation step in the initial sampling stage saves time and reduces the workload. The biomarker human chorionic gonadotropin (hCG) was used as the model analyte. The antibody-based paper samplers were prepared by functionalizing paper discs (6 mm) through a simple reaction using divinyl sulfone (DVS). After DVS activation, the paper discs were incubated with E27 hCG mAbs, followed by 0.05% tween/phosphate buffer saline to block the surface. After sample application and drying, the discs only needed to be washed before tryptic digestion and finally analysed on a nanoliquid chromatography-tandem mass spectrometry system. The finished DVS-mAbs samplers could selectively capture hCG (100 ng/mL) from human serum, with a recovery of 50%. Sample clean-up reduced the number of identified proteins from 132 to 82 before and after wash, respectively, with a 70% reduction in serum albumin signal while still retaining hCG on the sampler during the washing protocol. An evaluation of the samplers revealed excellent linearity (R2 = 0.9995) for hCG in serum with relative standard deviations below 15%. This work has presented the first ever reported paper samplers immobilized with antibodies utilizing DVS chemistry, showing promise in the future of paper-based sampling.

Parallel electromembrane extraction of peptides with monoterpene and medium-length fatty acid deep eutectic solvents.

BACKGROUND: Electromembrane extraction (EME) involves the process of mass transfer of charged analytes from an aqueous sample through an organic liquid membrane into an aqueous acceptor medium under the influence of an electrical field. Successful solvation of the analyte within the liquid membrane is of paramount importance and involves molecular interactions with the liquid membrane. In this comprehensive investigation, parallel EME was examined using a training set of 13 model peptides employing deep eutectic solvents as the liquid membrane. These deep eutectic solvents were formulated by mixing specific monoterpenes (thymol, menthol, camphor) with medium-chain fatty acids (1-octanoic acid and 1-decanoic acid). RESULTS: From an array of different liquid membrane compositions explored, it was revealed that the combination of camphor and 1-decanoic acid (in a 1:1 w/w ratio) with 2% di (2-ethylhexyl) phosphate (DEHP) delivered the most efficient extraction system. The solvation of the model peptides within this liquid membrane predominantly relied on ionic interactions between protonated basic functionalities and DEHP, along with hydrogen bond interactions between the deprotonated acid functionalities (hydrogen bond acceptor) and 1-decanoic acid (hydrogen bond donor). Selectivity was modulated by the pH of the sample and acceptor solutions, with a direct correlation to the polarity and net charge of the model peptides. The ionization of 1-decanoic acid in the interfacial region between the sample and liquid membrane emerged as an important factor influencing the selectivity. SIGNIFICANCE AND NOVELTY: Although parallel EME of peptides has been reported previously, the current liquid membrane provides an extraction system with sufficient stability for the first time. Selective extraction of peptides through EME holds substantial promise within the realm of next-generation environmentally-friendly sample preparation methodologies. The findings presented in this paper contribute significantly to our fundamental understanding of these processes, and may serve as an important reference for the development of future methods in this field.

Digging deeper into the field of the small cell lung cancer tumor marker ProGRP: a method for differentiation of its isoforms.

In this paper, we have used a newly developed immunocapture and LC-MS method to demonstrate for the first time the presence of protein isoforms 1 and 3 of the small cell lung cancer (SCLC) marker progastrin-releasing peptide (ProGRP) in sera. In addition, the method allows for indirect determination of the relative presence of the other known isoform of ProGRP, also known as ProGRP isoform 2. This new method is able to determine total ProGRP as a marker in sera at clinically relevant levels and to differentiate between isoforms at the low-pM level through combining selective sample preparation by immunoextraction, tryptic digestion, and separation followed by detection with LC-SRM-MS of the signature peptides, NLLGLIEAK (total ProGRP), LSAPGSQR (ProGRP isoform 1), and DLVDSLLQVLNVK (ProGRP isoform 3), with accuracies ≤ 25% for lower limit of quantification (LLOQ) and precisions ≤ 33%. By analyzing serum samples from four patients diagnosed with SCLC using the here described new and fully validated method, the ability is shown to both determine total ProGRP concentration and to differentiate between ProGRP isoforms 1 and 3 in one single run. Quantification of various ProGRP isoforms in one single run may be helpful for further understanding of the underlying biochemical processes in SCLC and differentiation of small cell lung cancer.