High Field Asymmetric Waveform Ion Mobility Spectrometry in Nontargeted Bottom-up Proteomics of Dried Blood Spots.

Despite the great potential of dried blood spots (DBS) as a source of endogenous proteins for biomarker discovery, the literature relating to nontargeted bottom-up proteomics of DBS is sparse, primarily due to the inherent complexity and very high dynamic range associated with these samples. Here, we present proof-of-concept results in which we have coupled high field asymmetric waveform ion mobility spectrometry (FAIMS) with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for nontargeted bottom-up proteomics of DBS with the aim of addressing these challenges. We, and others, have previously demonstrated the benefits of FAIMS more generally in proteomics including improved signal-to-noise and extended proteome coverage, and the aim of the current work was to extend those benefits specifically to DBS. The DBS samples were either extracted by the more traditional manual "punch and elute" approach or by an automated liquid surface extraction (LESA) approach prior to trypsin digestion. The resulting samples were analyzed by LC-MS/MS and LC-FAIMS-MS/MS analysis. The results show that the total number of proteins identified increased by ∼50% for the punch and elute samples and ∼45% for the LESA samples in the LC-FAIMS-MS/MS analysis. For both the punch and elute samples and the LESA samples, ∼30% of the total proteins identified were observed in both the LC-MS/MS and the LC-FAIMS-MS/MS data sets, illustrating the complementarity of the approaches. Overall, this work demonstrates the benefits of inclusion of FAIMS for nontargeted proteomics of DBS.

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.

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).

High-throughput analysis of drugs in biological fluids by desorption electrospray ionization mass spectrometry coupled with thin liquid membrane extraction.

Biological fluids such as urine, saliva and whole blood were analyzed for contents of drugs by a new combination of desorption electrospray ionization mass spectrometry (DESI-MS) and thin liquid membrane extraction (TLME). Analytes from the sample were extracted into a thin liquid membrane of hexadecane deposited on a porous Teflon membrane, from which they were subsequently analyzed directly by DESI. The total analysis time was 15 minutes for analysis of several samples with a potential analysis time of less than a minute per sample. Thanks to the pre-concentration and sample clean-up built into the method, methadone was detected in urine in full-scan mode with an LOD of 4 ng mL(-1), while amitriptyline, nortriptyline and pethidine showed LODs of 17 ng mL(-1). Quantification was possible for several basic drugs using one common internal standard, providing relative accuracies in the range of 10-30%. A reliability test was performed on 20 samples with methadone, amitriptyline, nortriptyline and pethidine in urine, showing that none of the samples having concentrations above the LOD were missed and no false positives were found. Diphenhydramine and one of its metabolites were detected in authentic samples of urine and saliva, and methadone was detected from a whole-blood sample spiked to a concentration of 100 ng mL(-1). The method has several advantages, such as extremely low price in consumables, the possibility of fast analysis of very crude biofluids such as whole blood and the potential for a very high sample throughput.

Matrix-Assisted Ionization and Tandem Mass Spectrometry Capabilities in Protein Biomarker Characterization-An Initial Study Using the Small Cell Lung Cancer Biomarker Progastrin Releasing Peptide as a Model Compound.

This initial study evaluates vacuum matrix-assisted ionization (vMAI) mass spectrometry (MS) for identification and determination of tryptic peptides from the biomarker protein progastrin releasing peptide (ProGRP). Similar peptides and charge states were observed as in liquid chromatography (LC) electrospray ionization (ESI) MS. The prolonged ion duration in vMAI with similar charge states as in ESI was advantageous for determining the MS/MS fragmentation conditions compared to MAI. It is assumed that the vacuum ionization conditions lower the detection limits of the experiment. This may be the reason vMAI combined with high resolution MS enabled detection of tryptic peptides from more digested proteins than MAI selected reaction monitoring MS. Additionally, MAI ion mobility spectrometry MS (MAI-IMS-MS) was evaluated for differentiation of intact protein isoforms, successfully enabling differentiation of the isoforms by drift time selection. Examples are both shown for model proteins bovine serum albumin, cytochrome C, and lysozyme and the clinically relevant small cell lung cancer protein biomarker ProGRP, which exists in three isoforms. Coupling with the vacuum ionization conditions using a dedicated vacuum-probe source MAI enables information to be extracted readily as with conventional approaches, just faster.

Biomonitoring of the benzene metabolite s-phenylmercapturic acid and the toluene metabolite s-benzylmercapturic acid in urine from firefighters.

The aim of this study was to determine firefighter's exposure to benzene and toluene during a fire drill by monitoring air benzene and toluene, and their corresponding urinary metabolites. A liquid chromatography mass spectrometry (LC-MS) method with a minimal of sample preparation steps was developed for the benzene metabolite s-phenylmercapturic acid (SPMA) and the toluene metabolite s-benzylmercapturic acid (SBMA) in urine. Urine samples and air samples were collected from nine firefighters during and after a fire drill in a non-environmentally refurbished house. Benzene and toluene were detected in the air samples with a median concentration of 15.5 ppm and 3.2 ppm, respectively. The metabolites SPMA and SBMA was also detected in all urine samples donated ≥ three hours after the fire drill with a median concentration of 0.6 µg/g creatinine and 5.9 µg/g creatinine, respectively. By our knowledge, this is the first study detecting SPMA in urine from firefighters.

Work Conditions and Practices in Norwegian Fire Departments From 1950 Until Today: A Survey on Factors Potentially Influencing Carcinogen Exposure.

BACKGROUND: Meta-analyses have shown firefighters to be at an increased risk of several cancer types. Occupational carcinogen exposure may explain these increased risks. This study aims to describe Norwegian fire departments' work conditions from 1950 until today, focusing on factors relevant for potential occupational carcinogen exposure. METHODS: With the help of a reference group, we developed a questionnaire on topics related to occupational exposure to carcinogens for the period 1950-2018. Selected Norwegian fire departments provided department-specific responses. RESULTS: Sixteen departments, providing fire services for 48% of the Norwegian population as of 2019 and mainly consisting of professional firefighters, responded to our questionnaire. The introduction of synthetic firefighting foams, more regular live fire training, the introduction of chemical diving, and a higher number of diesel-driven fire service vehicles were identified as changes thought to increase exposure to occupational carcinogens. Changes thought to decrease exposure included the switch from negative to positive pressure self-contained breathing apparatuses, the use of self-contained breathing apparatuses during all phases of firefighting, the use of ventilating fans during firefighting, increased attention to flammable materials used during live fire training, increased attention to handling and cleaning of turnout gear and other equipment, and installment of exhaust removal systems in apparatus bays. CONCLUSION: Norwegian fire departments' work conditions have seen several changes since 1950, and this could influence firefighters' occupational carcinogen exposure. A peak of carcinogen exposure may have occurred in the 1970s and 1980s before recent changes have reduced exposure.

Determination of the low-abundant protein biomarker hCG from dried matrix spots using immunocapture and nano liquid chromatography mass spectrometry.

Using LC-MS/MS for determination of low-abundance protein biomarkers from dried blood spots is challenging due to the combination of low biomarker levels (low pM-level) and small sample volumes (typically <50 µL). In the present paper it is demonstrated that use of state-of-the-art nano liquid chromatography triple quadrupole mass spectrometry in combination with immunoaffinity sample clean-up enable determination of the low abundance biomarker human chorionic gonadotropin (hCG) from four different biological matrices (whole blood, serum, plasma and urine) at its upper reference level (low pM). Detection limits for hCG was determined for all matrices from both commercially available non-soluble DBS sampling material (DMPK-C) and the water-soluble material carboxymethyl cellulose (CMC). The detection limits (S/N = 3) were ranging from 5.0 IU/L (14.5 pM; whole blood) to 10.5 IU/L (30.5 pM; urine) for DMPK-C and from 2.1 IU/L (6.1 pM; urine) to 6.4 IU/L (18.6 pM; plasma) for CMC. A brief evaluation was performed for both sampling materials using serum as matrix resulting in sufficient linearity (r2 ≥ 0.93, range 20-1000 IU/mL (58-2900 pM) for DMPK-C and 10-1000 IU/mL (29-2900 pM) for CMC), repeatability (RSD% = 13-31%) and accuracy (95-106%). To demonstrate the applicability of the method to real samples, a serum sample from a patient previously diagnosed with cancer was also analyzed using both sampling materials. The concentration levels found using the two materials were similar (5280±â€¯595 IU/L (15,312 ±â€¯1726 pM, n = 3) in the DMPK-C spot and 5060 ±â€¯430 IU/L (14,674 ±â€¯1247 pM, n = 3) in the CMC spot). All in all this demonstrated that the tools for determination of low abundance biomarkers at upper reference level from dried matrix spots now is available through a combination of immunoaffinity enrichment and state-of-the-art LC-MS/MS.

Volumetric absorptive MicroSampling vs. other blood sampling materials in LC-MS-based protein analysis - preliminary investigations.

The aim was to evaluate the performance of different microsampling materials in LC-MS-based protein analysis. The evaluated materials were the Volumetric Absorptive MicroSampling (VAMS) device, the pure cellulose sampling material (DMPK-C) and the water-soluble material (carboxymethyl cellulose, CMC), with the main emphasis on VAMS. Six proteins with different physicochemical properties were used as model proteins. A quick and generic sample preparation consisting of extraction/dissolution of the blood spot, tryptic digestion and subsequent matrix precipitation was applied prior to analysis. The recovery from VAMS, compared to DMPK-C, was dependent on the protein analyte: Lower recovery compared to DMPK-C was seen for ß-lactoglobulin and myoglobin (74% and 80%, respectively) while higher recovery compared to DMPK-C was seen for cytochrome c and albumin (149% and 197%, respectively). The recovery from CMC was comparable to the recovery from DMPK-C for all proteins except for cytochrome c (76%). Hematocrit bias was evaluated for DMPK-C and VAMS, and the blood hematocrit influenced the protein analysis from both the materials. A preliminary evaluation was performed for VAMS: The correlation coefficient (R2 ≥ 0.983), the accuracy (71-101%) and the precision (RSD ≤ 20%) were determined for blood spiked with the six model proteins.

Evaluation of water-soluble DBS for small proteins: a conceptual study using insulin as a model analyte.

AIM: Water-soluble sampling materials for DBS have been introduced to solve some of the common challenges of DBS. METHODOLOGY: Carboxymethyl cellulose (CMC) as water-soluble material was evaluated for small proteins using insulin as model analyte. 15 µl of whole blood was deposited and dried on a sheet of CMC prior to dissolvation of the whole spot, matrix precipitation with acetonitrile and LC-MS/MS analysis. RESULTS: CMC was shown to promote matrix precipitation resulting in cleaner extracts than precipitation without CMC present. The recovery of insulin from the spot was 68 ± 4%, and the spotted samples were stable for at least 1 week in room temperature. CONCLUSION: Water-soluble DBS showed promising performance also in analysis of small proteins.