We greatly acknowledge Prof. Elisabeth Paus at the Norwegian Radium Hospital (Oslo University Hospital, Oslo, Norway) for providing the ProGRP standard and the anti-ProGRP monoclonal antibody, M18. The publication fee was covered by a grant from Apoteker Harald Conrad Thaulows legat. Trine Gr&#248;nhaug Halvorsen and L&#233;on Reubsaet are partners in the National Network of Advanced Proteomics Infrastructure (NAPI) consortium, which is funded by the Research Council of Norway INFRASTRUKTUR-program (project number: 295910).

Serum from healthy blood donors was used for the preparation of standard solutions. The use of serum from healthy blood donors was performed in strict accordance with Norwegian law. Informed consent was obtained from all subjects. Serum samples were analyzed using methods in accordance with relevant guidelines and regulations. The protocol described is a modified version of the method described in previous work14. An overview of the composition of buffers and solutions and how to prepare them can be found in Supplemental Table S1, while the Table of Materials contains materials, equipment, and reagents used in this protocol.
1. Preparation of antibody-coated magnetic beads
<ol>
	<li>Calculate the amount of antibody necessary to prepare the desired number of beads. Use 1 mg of antibody per 50 mg of magnetic beads. 
	NOTE: Typically, 20 &#181;L of a 20 mg/mL bead suspension is used per sample in subsequent experiments (see step 5.1).</li>
	<li>Calculate the antibody volume necessary to prepare the desired number of beads. 
	NOTE: The antibody volume is dependent on antibody concentration and needs to be calculated for each antibody.</li>
	<li>Transfer the desired volume of antibody solution to a 5 mL low-protein-binding microcentrifuge tube. As an example, if the antibody solution contains 1 mg/mL antibody, use 0.4 mL to prepare 1.0 mL of bead suspension (20 mg beads/mL with 1 mg antibody/50 mg beads). 
	NOTE: The antibodies should be in an amine-free buffer. Use low-protein-binding microcentrifuge tubes for all solutions containing proteins to avoid analyte loss due to adsorption.</li>
	<li>Add a small magnetic stirring bar to the antibody solution and adjust the pH to 2.5 under continuous stirring. Use a pH meter with a micro electrode to measure the pH and adjust the pH by stepwise addition of defined volumes of 1.0 M HCl (start by adding 10 &#181;L portions of 1.0 M HCl and reduce the volume as the pH approaches 2.5). Record the total volume of 1.0 M HCl necessary to adjust the pH to 2.5.</li>
	<li>Remove the micro electrode and incubate the acidified antibody on ice on a magnetic stirrer for 1 h. 
	NOTE: Acid treatment of the antibody is performed to promote the correct orientation of the antibodies on the bead. The concentration of HCl may be reduced to 0.5 M or 0.2 M HCl, depending on the buffer concentration in the antibody solution.</li>
	<li>Neutralize the antibody solution. Use a pH meter with a micro electrode to measure the pH and adjust the pH to 7 by stepwise addition of defined volumes of 1.0 M NaOH (start with a 10 &#181;L portion and reduce the volume as the pH approaches 7). Record the total volume of 1.0 M NaOH added. 
	NOTE: The concentration of NaOH should be the same as the HCl concentration used in step 1.4. NaOH is highly corrosive; use protective equipment, including gloves and appropriate eye protection.</li>
	<li>Calculate the desired volume of tosyl activated magnetic beads to be used. 
	NOTE: Typically, it is recommended to use 20 mg beads/mL when performing small-scale coupling. A minimum of 5 mg of beads should be used.</li>
	<li>Mix the magnetic bead suspension thoroughly on a vortex mixer and withdraw a volume containing the desired amount of bead suspension. As an example, to prepare 1 mL of bead suspension, withdraw a volume containing 20 mg of beads (667 &#181;L if the concentration of beads is 30 mg/mL).</li>
	<li>Place the bead suspension on a magnetic rack for 1 min and remove the supernatant. Wash the beads 2x with equal volume of Type 1 H2O (667 &#181;L if a 30 mg/mL bead solution is used to prepare 1 mL of 20 mg/mL antibody-coated beads). Mix using a vortex mixer after each addition of wash solution and place on the magnetic rack for 1 min prior to removing the supernatant.</li>
	<li>Add the following to the magnetic beads: acid-treated antibody, 0.5 M borate buffer (pH 9.5) (1/5 of total volume as the final concentration should be 0.1 M; this equals 0.2 mL to prepare 1 mL of bead suspension), and coupling buffer for antibody coupling (to prepare 1 mL of antibody-coated magnetic beads, the volume of coupling buffer should equal 1 mL - volume of acid treated antibody - 0.2 mL borate buffer). Mix using a vortex mixer. 
	NOTE: The volume of acid-treated antibody equals the volume of antibody solution (0.4 mL in preparation of 1 mL of bead suspension using a 1 mg/mL antibody solution) + the volume of 1.0 M HCl used to adjust the pH to 2.5 + the volume of 1.0 M NaOH used to adjust the pH to 7). 
	NOTE: The composition of 0.5 M borate buffer (pH 9.5) and coupling buffer for antibody coupling can be found in Supplemental Table S1.</li>
	<li>Rotate at ambient temperature overnight using an end-over-end sample mixer, preferably with reciprocal rotation and vibration.</li>
	<li>Centrifuge for 10 min at 239 &#215; g. Place the tube on the magnet for 2 min and remove the supernatant.</li>
	<li>Wash the beads 2 x 2 h and once overnight by rotating in storage buffer for antibody beads&#160;at ambient temperature using an end-over-end sample mixer. Use the same volume as the total volume in step 1.10. Place the tube on the magnet for 1 min and remove the supernatant between each wash. 
	&#8203;NOTE: The composition of storage buffer for antibody beads can be found in Supplemental Table S1.</li>
	<li>Store in the desired buffer (e.g., the same buffer as in step 1.13) using the desired stock concentration of beads (typically 20 mg/mL). Store in the fridge.</li>
</ol>
2. Preparation of 2 mL of trypsin immobilized beads (20 mg/mL beads)
<ol>
	<li>Add 20 mL of 1 mM HCl to 2 mL of NHS-activated agarose beads to wash the beads.</li>
	<li>Mix and centrifuge at 2,655 &#215; g for 5 min. Remove the supernatant.</li>
	<li>Add 20 mL of 0.1 M phosphate buffer (pH 7.8). Mix using a vortex mixer and centrifuge at 2,655 &#215; g for 5 min. Remove the supernatant. 
	NOTE: The composition of 0.1 M phosphate buffer (pH 7.8) can be found in Supplemental Table S1.</li>
	<li>Add 2 mL of 20 mg/mL trypsin (dissolved in cold coupling buffer for trypsin coupling). 
	NOTE: The composition of coupling buffer for trypsin coupling can be found in Supplemental Table S1. Store the buffer in the fridge.</li>
	<li>Incubate for 25 min at 22 &#176;C and 1,100 rpm using a temperature-controlled mixer. Centrifuge at 2,655 &#215; g for 5 min. Remove the supernatant.</li>
	<li>Add 2 mL of modification buffer for the preparation of trypsin beads and incubate for 20 min at 22 &#176;C and 1,100 rpm using a temperature-controlled mixer. Centrifuge at 2,655 &#215; g for 5 min. Remove the supernatant. 
	NOTE: The composition of the modification buffer for the preparation of trypsin beads can be found in Supplemental Table S1. Store the buffer in the fridge.</li>
	<li>Add 10 mL of blocking buffer for the preparation of trypsin beads and incubate for 10 min at 22 &#176;C and 1,100 rpm using a temperature-controlled mixer. Centrifuge at 2,655 &#215; g for 5 min. Remove the supernatant. 
	NOTE: The composition of blocking buffer for the preparation of trypsin beads can be found in Supplemental Table S1. Store the buffer in the fridge.</li>
	<li>Add 2 mL of storage buffer, mix using a vortex mixer, and store in the fridge until use. 
	&#8203;NOTE: The composition of storage buffer for trypsin beads can be found in Supplemental Table S1. Store the buffer in the fridge.</li>
</ol>
3. DSS/VAMS&#160;sampling and subsequent extraction of dried serum
<ol>
	<li>Prepare standards by spiking the serum with ProGRP (or the protein of interest) at the appropriate level. Keep the spiking volume &#8804; 1% of the total volume. 
	&#8203;NOTE: Here, a stock solution of 295 &#181;g/mL ProGRP in water was used for the preparation of spiked serum standards.</li>
	<li>Mix the standards on a vortex mixer.</li>
	<li>Apply 10 &#181;L of serum (spiked standards) onto DBS cards or allow 10 &#181;L of serum to be collected by VAMS (10 &#181;L) using the manufacturer&#39;s guidelines. For DSS, make sure that the spot is within the dotted circle.</li>
	<li>Allow to air dry at ambient temperature for at least 2 h.</li>
	<li>Cut out the whole spot and transfer it to a 2 mL low-protein-binding microcentrifuge tube or remove the VAMS from the holder and place it in a 2 mL low-protein-binding microcentrifuge tube.</li>
	<li>Add 1,000 &#181;L of 100 mM ammonium bicarbonate (ABC) solution. Extract the dried serum from the spot/VAMS for 1 h at 22 &#176;C using a temperature-controlled mixer at 1,000 rpm.</li>
	<li>Transfer the extract to a new 1.5 mL low-protein-binding microcentrifuge tube for tryptic proteolysis.</li>
</ol>
4. Digestion of DSS/VAMS extracts
<ol>
	<li>Use 30 &#181;L of trypsin beads (as prepared in section 2) per sample. Transfer a volume containing trypsin beads for all samples to a new 1.5 mL low-protein-binding microcentrifuge tube, centrifuge at 2,655 &#215; g for 5 min, and remove the supernatant.</li>
	<li>Wash the trypsin beads 2x with 1 mL of cold 50 mM ABC, before resuspending in the same volume that was pipetted out. Centrifuge at 2,655 &#215; g for 5 min and remove the supernatant between steps.</li>
	<li>Add 30 &#181;L of washed trypsin beads to each DSS/VAMS extract to initiate digestion.</li>
	<li>Incubate for 2 h at 37 &#176;C and 1,000 rpm using a temperature-controlled mixer or similar. Centrifuge at 2,655 &#215; g for 5 min and transfer the supernatant (not beads) to a new 2.0 mL low-protein-binding microcentrifuge tube.</li>
	<li>Add 25 &#181;L of 14 ng/mL internal standard (IS) solution containing the stable isotope-labeled (SIL) peptide ALGNQQPSWDSEDSSNF[K_13C6_15N2] in 100 mM ABC solution.</li>
</ol>
5. Capture of proteotypic ProGRP peptide using antibody-coated magnetic beads
<ol>
	<li>Use 20 &#181;L of antibody coated beads (20 mg/mL as prepared in section 1) per extraction. Transfer all antibody-coated beads to a new 1.5 mL low-protein-binding microcentrifuge tube, place on the magnet for 1 min, and remove the storage buffer.</li>
	<li>Wash the magnetic antibody-coated beads with 1 mL of phosphate-buffered saline (PBS), pH 7.4, containing 0.05% polysorbate 20, and 2 x 1 mL of PBS before resuspending in the same volume that was pipetted out. Mix on a vortex mixer and place on the magnet for 1 min between buffer exchanges. 
	NOTE: PBS contains 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 1.8 mM KH2PO4. It is recommended to prepare the solution at a 10x concentration for increased stability. For the composition of 100 mL of 10x PBS, see Supplemental Table S1. Dilution of the 10x PBS 10x will achieve a pH of ~7.4.</li>
	<li>Add 20 &#181;L of washed magnetic bead suspension to each low-protein-binding microcentrifuge tube containing a digested DSS/VAMS extract and IS. Perform immunoextraction for 1 h using an end-over-end sample mixer at ambient temperature.</li>
	<li>Wash the magnetic beads using the following solutions: 500 &#181;L of PBS with 0.05% (v/v) polysorbate 20, 400 &#181;L of PBS, 300 &#181;L of 10 mM Tris HCl (pH 7.4), and 300 &#181;L of 100 mM ABC.
	<ol>
		<li>After the addition of each wash solution, remove the low-protein-binding microcentrifuge tube with beads and wash solution from the magnet rack and invert carefully until homogeneous. Then, place the low-protein-binding microcentrifuge tube on the magnet for 30 s, invert for 30 s, and place on the magnet for 1 min. Remove the wash solution.</li>
		<li>Spin down the remaining suspension using a microcentrifuge. Place on the magnet for 1 min and remove the remaining wash solution.</li>
	</ol>
	</li>
	<li>Add 15 &#181;L of 2% (v/v) formic acid in Type 1 H2O to each sample and incubate for 5 min at 22 &#176;C and 1,000 rpm using a temperature-controlled mixer, or similar, to elute the captured peptides. Place on the magnet for 1 min and transfer the eluate to a new 1.5 mL low-protein-binding microcentrifuge tube. Repeat once and transfer the second eluate to the same low-protein-binding microcentrifuge tube as the first eluate.</li>
	<li>Add 20 &#181;L of 100 mM ABC to each eluate (total volume of 50 &#181;L).</li>
	<li>Centrifuge (microcentrifuge) and transfer 40 &#181;L of the eluate to micro inserts for high-performance liquid chromatography (HPLC) vials.</li>
</ol>
6. Analysis by LC-MS/MS
<ol>
	<li>Use a micro LC triple quadrupole MS with electrospray ionization for high robustness.</li>
	<li>Prepare the LC-MS/MS system for the analysis by purging the pumps with mobile phase A (20 mM FA in H2O and MeCN, 95:5 v/v) and B (20 mM FA in H2O and MeCN, 5:95 v/v).</li>
	<li>Insert the analytical column (C18, 50 mm x 1 mm inner diameter [ID], 3 &#181;m particles).</li>
	<li>Continue to prepare the instrument for analysis by following the start-up procedures for the specific instrument.</li>
	<li>In the instrument software, set the column oven temperature to 25 &#176;C and the flow rate to 50 &#181;L/min (100% mobile phase A).</li>
	<li>Equilibrate the column with mobile phase A for at least 15 min.</li>
	<li>Place the samples in the autosampler.</li>
	<li>Prepare the instrument method for analysis of the ProGRP-specific, tryptic peptide ALGNQQPSWDSEDSSNFK, and its IS. 
	NOTE: Several method parameters are instrument-specific and must be optimized on the specific instrument used. Details of the method parameters used here are described in steps 6.9 to 6.11.</li>
	<li>For the gradient program in LC, use the following settings: flow rate: 50 &#181;L/min; from time 0.0 to 3.0 min: 100% mobile phase A; from time 3.0 to 18.0 min: run a linear gradient from 0% to 50% mobile phase B; from time 18.0 to 18.1 min: increase mobile phase B from 50% to 100%; from time 18.1 to 20.0 min: 100% mobile phase B; from time 20.0 to 20.1 min: decrease mobile phase B from 100% to 0%; from time 20.1 to 30 min: 100% mobile phase A to re-equilibrate the column (use at least 10 column volumes).</li>
	<li>For MS/MS settings, run MS/MS in positive mode using instrument settings that ensure efficient ionization. To follow this protocol, use a spray voltage of +4,000 V, a heated capillary temperature of 270 &#176;C, nitrogen as sheet gas (5 arbitrary units), and argon (2 mTorr) for collision-induced dissociation (CID) fragmentation. If the instrument allows, direct the LC flow to waste the first 2 min (0-2 min) and the last 1 min (29-30 min) of the run, instead of into the MS.</li>
	<li>Use selected reaction monitoring (SRM) transitions for (A) the signature peptide (ALGNQQPSWDSEDSSNFK): 1,005.45&#8594;913.3, 1,005.45&#8594;1,028.3, and 1,005.45&#8594;1,398.5, and (B) the IS SIL peptide (ALGNQQPSWDSEDSSNF[K_13C6_15N2]): 1,009.45&#8594;921.3, 1,009.45&#8594;1,036.3, and 1,009.45&#8594;1,406.5. Use optimized collision energy (35 V in this protocol) for all monitored transitions.</li>
	<li>Prepare a sequence containing the samples to be run using the software available for your LC-MS/MS system. Set the injection volume to 10 &#181;L. 
	NOTE: Make sure to add blank samples and quality control samples as needed.</li>
	<li>Press &#34;Run sequence&#34; in the instrument software to start the sequence.</li>
	<li>Monitor the peak area of the affinity-captured, ProGRP-specific, tryptic peptide ALGNQQPSWDSEDSSNFK, and its IS SIL peptide. 
	NOTE: Selection and confirmation of the signature peptide, ALGNQQPSWDSEDSSNFK, is described elsewhere14.</li>
</ol>

<ol>
	<li>Bang, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fresenius.">Fresenius.</a> Zeitschrift f&#252;r analytische Chemie. 52 (7), 521-523 (1913).</li><li>Guthrie, R., Susi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+phenylalanine+method+for+detecting+phenylketonuria+in+large+populations+of+newborn+infants.">A simple phenylalanine method for detecting phenylketonuria in large populations of newborn infants.</a> Pediatrics. 32 (3), 338-343 (1963).</li><li>Laurell, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+screening+test+for+&#945;1-antitrypsin+deficiency.">A screening test for &#945;1-antitrypsin deficiency.</a> Scandinavian Journal of Clinical and Laboratory Investigation. 29 (3), 247-248 (1972).</li><li>Thielmann, K., Aquino, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+blood+samples+dried+and+stored+on+filter+paper+as+substrate+for+the+electrophoretic+separation+on+hemoglobin+S+from+hemoglobin+A.+A+screening+procedure.">Whole blood samples dried and stored on filter paper as substrate for the electrophoretic separation on hemoglobin S from hemoglobin A. A screening procedure.</a> Clinica Chimica Acta. 35 (1), 237-238 (1971).</li><li>Wada, Y., Fujita, T., Kidoguchi, K., Hayashi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+hemoglobin+variants+in+80,000+Japanese+neonates:+High+prevalence+of+Hb+F+Yamaguchi+(A&#947;T+80+Asp&#8594;Asn).">Fetal hemoglobin variants in 80,000 Japanese neonates: High prevalence of Hb F Yamaguchi (A&#947;T 80 Asp&#8594;Asn).</a> Human Genetics. 72 (3), 196-202 (1986).</li><li>Halvorsen, T. G., McKitterick, N., Kish, M., Reubsaet, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+capture+in+bottom-up+protein+analysis-overview+of+current+status+of+proteolytic+peptide+capture+using+antibodies+and+molecularly+imprinted+polymers.">Affinity capture in bottom-up protein analysis-overview of current status of proteolytic peptide capture using antibodies and molecularly imprinted polymers.</a> Analytica Chimica Acta. 1182, 338714 (2021).</li><li>Halvorsen, T. 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The authors have no conflicts of interest to disclose.

The described protocol contains information about how to conduct several important steps in the analysis of low-abundance biomarkers from dried microsamples (DSS and VAMS), including the preparation of trypsin beads and antibody-coated magnetic beads. Based on previous experience, we always treat the antibody with acid prior to bead immobilization to improve the orientation of the antibodies15. 
One of the critical steps in this procedure is the selection of the most suitable microsampling format. First, one must consider if the analyte in question can be determined from whole blood, or if the concentration is influenced by the blood cells and has to be determined in serum or plasma (as for the model analyte, ProGRP).
Both paper- and polymer-based approaches have advantages and limitations; for ProGRP, VAMS provides a clear advantage with respect to analyte recovery after extraction from the sampler. This can, however, probably be optimized using a different extraction solution for the DSS samples. Nevertheless, this potential interaction between the analyte and the sampling material is important to take into account as it might result in increased analytical variation and higher detection limits. As the IS used is an SIL peptide and first added after the digestion, IS corrects for the steps following digestion (e.g., affinity extraction and LC-MS/MS analysis). IS correction is not possible for extraction from DSS/VAMS and the digestion step.
Two types of beads are used in the procedure: trypsin beads for digestion after extraction of the serum sample from the sampler, and antibody-coated magnetic beads for capture of the proteotypic peptide after digestion. A major reason for using trypsin beads, in addition to speeding up the digestion, is to minimize residual trypsin activity in the sample during affinity capture. This is important to avoid tryptic proteolysis of the mAb during affinity capture. 
Agarose beads were used for preparation of the trypsin beads, while magnetic beads were used for preparation of the antibody-coated beads. Agarose beads are less expensive than magnetic beads but have a limitation that separation of the beads from the solution requires centrifugation. This makes the separation of beads and supernatant less efficient than when using magnetic beads. In addition, automation of the workflow is difficult using agarose beads. However, magnetic NHS-activated beads are available and can be used for a more streamlined and automatable sample preparation workflow. 
Microsampling is an important trend in the bioanalysis of both drugs and biomarkers. One challenge with the current approach is the limited amount of sample volume (10 &#181;L), which may be of particular importance in the determination of very-low-abundance analytes such as ProGRP (pg/mL-low ng/mL level). However, this challenge may be circumvented using state-of-the-art analytical equipment. For these low-abundance analytes, the choice of sample preparation is crucial, and selective sample cleanup through antibody-based affinity capture is most often needed. As peptide capture has been shown to provide cleaner extracts and lower detection limits than protein capture (using the same antibody)14, the present method focuses on this approach in combination with microsampling. Another advantage of the peptide capture approach is that the IS SIL peptide also corrects for the affinity capture step. 
In this work, an antibody targeting a protein was used for peptide capture. This is an advantage as the availability of off-the-shelf antibodies targeting proteins are higher than off-the-shelf antibodies targeting proteotypic peptides. However, for an anti-protein antibody to efficiently capture a proteotypic peptide, the epitope needs to be intact after protein digestion. In addition, for many antibodies, the exact epitope is not known, making the search for an anti-protein antibody tedious. This limits the number of available anti-protein antibodies applicable for peptide capture. The described procedure is demonstrated using serum as the matrix and ProGRP as the target analyte. The procedure is intended to be applicable to other matrixes and other target analytes. Instead of using a commercially available anti-protein antibody for the affinity capture of the proteotypic peptide, it is possible to also use custom-made, anti-peptide antibodies. The cleanup efficiency of peptide capture compared to protein capture is illustrated in Figure 5. By exchanging the agarose beads used for the preparation of trypsin beads with magnetic beads, the procedure should also be compatible with robotic sample preparation work stations on the market.

Microsampling has been around for more than 100 years since Ivar Bang described glucose monitoring from DBSs in 19131. After Guthrie and Susi introduced DBSs in 1963 for the determination of phenylalanine in newborns2, the technique has become increasingly widespread. The first reports of DBSs for the sampling and storage of proteins were made in the early 1970s3,4, and a decade later, in the 1980s, we found the first report of mass spectrometry (MS) for the determination of proteins from DBSs5. Despite this early introduction, it was not until after the turn of the century that MS determination of proteins from DBSs and other microsampling techniques became more widespread.
In a clinical context, it is of interest to determine proteins in the diagnosis and follow-up of diseases, as well as for treatment monitoring and doping purposes. This targeted determination of protein analytes by MS from small amounts of dried samples is still challenging, and often requires extensive sample preparation prior to analysis.
Targeted quantitative determination of proteins by MS is commonly performed by applying the bottom-up approach, digesting the proteins to peptides prior to analysis. This procedure produces a myriad of peptides, which makes direct analysis of the digested biological sample challenging. A way of circumventing this is to apply a selective affinity cleanup step upfront of MS analysis either before or after the digestion6,7,8. In this way, the protein of interest (or its proteotypic peptide, if the affinity capture step is performed after the digestion) is selectively isolated from the sample matrix prior to analysis, providing lower detection limits9.
Microsampling using DBS cards has certain advantages compared to conventional blood samples, including low sample volume, less invasive sampling, and increased storage stability. However, the sample matrix is different and can introduce other challenges in the analysis (e.g., dried vs. liquid sample matrix and capillary blood vs. serum or plasma)10,11. Another challenge that is observed with DBSs is the so-called hematocrit effect, where the blood hematocrit affects the sample volume further processed for analysis, and hence introduces interindividual variability in the analysis12. Newer microsampling units, such as VAMS introduced in 201413, address this issue by collecting a fixed volume of blood instead of a blood drop.
This protocol describes a setup for the analysis of low-abundance biomarkers from dried microsamples. After elution, the dried sample is digested and, subsequently, the proteotypic peptide is isolated by peptide affinity capture. The model analyte is the SCLC biomarker ProGRP. As ProGRP cannot be determined reliably from whole blood, serum was used as the sample matrix. Representative results from both DSSs and serum samples collected using VAMS are shown.

<table><tbody><tr><td>Acetic acid N-hydroxysuccinimide ester</td><td>Carbosynt (Staad, Switzerland)</td><td>FA33719</td><td>Store in freezer below -20 &amp;deg;C</td></tr><tr><td>ALGNQQPSWDSEDSSNF[K_13C6&lt;br/&gt; _15N2] (&amp;ge; 95%)</td><td>Innovagen (Lund, Sweden)</td><td>Not applicable</td><td>Store in freezer below -20 &amp;deg;C</td></tr><tr><td>Ammonium bicarbonate BioUltra (&amp;ge; 99.5% )</td><td>Sigma Aldrich (St. Louis, MO, USA)</td><td>09830-500G</td><td></td></tr><tr><td>Aquasil C18 column, 3 &amp;micro;m, 50 mm x 1 mm</td><td>Thermo scientific (Waltham, MA, USA)</td><td>77503-051030</td><td>Analytical column compatible with 100% aqueous mobile phase</td></tr><tr><td>Benzamidine (&amp;ge; 95.0% )</td><td>Sigma Aldrich (St. Louis, MO, USA)</td><td>12072</td><td>Store in fridge at 2-6 &amp;deg;C</td></tr><tr><td>Calsium chloride dihydrate&amp;nbsp; (&amp;ge; 99% )</td><td>Sigma Aldrich (St. Louis, MO, USA)</td><td>223506-500G</td><td></td></tr><tr><td>Centrifuge 5804</td><td>Eppendorf (Hamburg, Tyskland)</td><td>5804000010</td><td></td></tr><tr><td>Cloned ProGRP isoform 1</td><td>Radium hospital, Oslo University Hospital (Oslo, Norway)</td><td>Not applicable</td><td>Store in freezer below -20 &amp;deg;C</td></tr><tr><td>Disodium hydrogenphosphate dihydrate (pro analysis)&amp;nbsp;</td><td>Sigma Aldrich (St. Louis, MO, USA)</td><td>30435-500G</td><td></td></tr><tr><td>Disodium hydrogenphosphate dodecahydrate (pro analysis)&amp;nbsp;</td><td>Merck (Darmstadt, Tyskland)</td><td>1.06579.0500</td><td></td></tr><tr><td>Dynabeads M-280 tosylactivated 10 mL</td><td>Invitrogen (Carlsbad, USA)</td><td>14204</td><td>Store in fridge at 2-6 &amp;deg;C</td></tr><tr><td>DynaMag-2</td><td>Invitrogen (Carlsbad, USA)</td><td>123-21D</td><td></td></tr><tr><td>Ethanolamine (pro analysis, &amp;ge; 99%)</td><td>Sigma Aldrich (St. Louis, MO, USA)</td><td>#02400</td><td></td></tr><tr><td>Formic acid (&amp;ge; 99% ) for LC-MS</td><td>VWR International (Radnor, PA, USA)</td><td>84865.260</td><td></td></tr><tr><td>FTA DMPK-C cellulose card</td><td>Whatman (Kent, UK)</td><td>WB129243</td><td>DBS card</td></tr><tr><td>HPLC vials, clear glass, 1.5 mL, 32 x 11.6 mm, Clean Pack</td><td>Nerliens Meszansky (Oslo, Norge)</td><td>LPP 11 09 0519</td><td></td></tr><tr><td>Hulamixer sample mixer</td><td>Invitrogen (Carlsbad, USA)</td><td>101561503016</td><td>Sample mixer with end-over-end mixing and reciprocal rotation and vibration</td></tr><tr><td>Human serum from healty blood donors</td><td>Bloodbank, Ullev&amp;aring;l, Oslo University Hospital (Oslo, Norway)</td><td>Not applicable</td><td>Store in freezer below -20 &amp;deg;C</td></tr><tr><td>Hydrochloric acid fuming 37% (Emsure for analysis)</td><td>Merck (Darmstadt, Tyskland)</td><td>1.00317.1000</td><td></td></tr><tr><td>LC-MS/MS system: Ultimate 3000 system (Autosampler, WPS-3000TRS; Micropump, LPG-3400M; Flow manager, FLM-3300, MIC, 1X2P-10P) and TSQ Quantum access. Controlled by Xcalibur 2.2 SP1.48</td><td>Thermo scientific (Waltham, MA, USA)</td><td>Not applicable</td><td></td></tr><tr><td>LiChrosolv Acetonitrile hypergrade for LC-MS</td><td>Merck (Darmstadt, Tyskland)</td><td>1.00029.2500</td><td></td></tr><tr><td>LL Biotrode, Combined glass electrode</td><td>Metrohm (Herisau, Sveits)</td><td>6.0224.100</td><td></td></tr><tr><td>Magnetic stirrer, Type M10</td><td>Franz Morat KG (Eisenbach, Germany)</td><td>10236</td><td></td></tr><tr><td>Micro inserts, glass (31 x 6 mm, 0.1 mL)</td><td>VWR International (Radnor, PA, USA)</td><td>548-0006</td><td></td></tr><tr><td>MilliQ integral 3 with Q-POD</td><td>Merck Millipore (Molsheim, France)</td><td>ZRXQ003T0</td><td>For production of Type 1 water</td></tr><tr><td>monoclonal antibody M18</td><td>Radium hospital, Oslo University Hospital (Oslo, Norway)</td><td>Not applicable</td><td>Store in fridge at 2-6 &amp;deg;C</td></tr><tr><td>Neoteryx Mitra microsampler (10 &amp;mu;L) 4 sampler Clamshell</td><td>Fisher Scientific (Waltham, MA, USA)</td><td>NC1382947</td><td></td></tr><tr><td>NHS-activated sepharose beads 4 fast flow</td><td>Sigma Aldrich (St. Louis, MO, USA)</td><td>GE17-0906-01</td><td>Agarose beads, store in fridge at 2-6 &amp;deg;C</td></tr><tr><td>Optifit, Refill pipet tips, 10 mL</td><td>Sartorius Biohit (Helsinki, Finland)</td><td>613-2911</td><td></td></tr><tr><td>Optifit, Refill pipet tips, 10 &amp;mu;L</td><td>Sartorius Biohit (Helsinki, Finland)</td><td>790012</td><td></td></tr><tr><td>Optifit, Refill pipet tips, 1,000 &amp;mu;L</td><td>Sartorius Biohit (Helsinki, Finland)</td><td>791002</td><td></td></tr><tr><td>Optifit, Refill pipet tips, 200 &amp;mu;L</td><td>Sartorius Biohit (Helsinki, Finland)</td><td>790202</td><td></td></tr><tr><td>pH glass electrode</td><td>Metrohm (Herisau, Sveits)</td><td>6.0233.100</td><td></td></tr><tr><td>pH meter 744</td><td>Metrohm (Herisau, Sveits)</td><td>8.744.1003</td><td></td></tr><tr><td>Pipet 10 mL</td><td>Sartorius Biohit (Helsinki, Finland)</td><td>725090</td><td></td></tr><tr><td>Pipet m10 &amp;micro;L&amp;nbsp;</td><td>Sartorius Biohit (Helsinki, Finland)</td><td>725020</td><td></td></tr><tr><td>Pipet m100 &amp;micro;L</td><td>Sartorius Biohit (Helsinki, Finland)</td><td>725050</td><td></td></tr><tr><td>Pipet m1,000 &amp;micro;L</td><td>Sartorius Biohit (Helsinki, Finland)</td><td>725070</td><td></td></tr><tr><td>Pipet m20 &amp;micro;L</td><td>Sartorius Biohit (Helsinki, Finland)</td><td>725030</td><td></td></tr><tr><td>Potassium chloride (KCl &amp;ge; 99.9%)</td><td>Sigma Aldrich (St. Louis, MO, USA)</td><td>P-3911</td><td></td></tr><tr><td>Potassium dihydrogenphosphate (pro analysis)</td><td>Merck (Darmstadt, Tyskland)</td><td>1.04873.0250</td><td></td></tr><tr><td>Protein LoBind Eppendorf tube 0.5 mL</td><td>Eppendorf (Hamburg, Tyskland)</td><td>525-0133 (0030 108.094)</td><td></td></tr><tr><td>Protein LoBind Eppendorf tube 1.5 mL</td><td>Eppendorf (Hamburg, Tyskland)</td><td>525-0132 (0030 108.116)</td><td></td></tr><tr><td>Protein LoBind Eppendorf tube 2.0 mL</td><td>Eppendorf (Hamburg, Tyskland)</td><td>525-0134 (0030 108.450)</td><td></td></tr><tr><td>Protein LoBind Eppendorf tube 5.0 mL</td><td>Eppendorf (Hamburg, Tyskland)</td><td>525-0792 (0030108.302)</td><td></td></tr><tr><td>Scissors</td><td>Sigma Aldrich (St. Louis, MO, USA)</td><td>Z186716-1EA</td><td></td></tr><tr><td>Sodium azide (BioUltra; &amp;ge; 99.5% )</td><td>Sigma Aldrich (St. Louis, MO, USA)</td><td>71289-5G</td><td></td></tr><tr><td>Sodium chloride (for analysis)</td><td>Merck (Darmstadt, Tyskland)</td><td>1.06404.1000</td><td></td></tr><tr><td>Sodium dihydrogenphosphate monohydrate (pro analysis)</td><td>Merck (Darmstadt, Tyskland)</td><td>1.06346.0500</td><td></td></tr><tr><td>Sodium hydroxide (AnalaR NORMAPUR)</td><td>VWR International (Radnor, PA, USA)</td><td>28244.295</td><td></td></tr><tr><td>Sodium tetraborate decahydrate (&amp;ge; 99. %)</td><td>Sigma Aldrich (St. Louis, MO, USA)</td><td>S9640-500G</td><td></td></tr><tr><td>Spectrafuge Mini Centrifuge</td><td>LABNET International (Edison, NJ, USA)</td><td>C1301</td><td></td></tr><tr><td>Stirring magnet, 25 mm x 6 mm &amp;Oslash;, circular</td><td>Leybold (Cologne, Germany)</td><td>666&amp;nbsp;851</td><td></td></tr><tr><td>Stuart&amp;nbsp;Scientific SA8 vortex mixer</td><td>Stuart (Staffordshire, UK)</td><td>Z648531-1EA</td><td></td></tr><tr><td>SuperClear centrifuge tubes (15 mL)</td><td>VWR International (Radnor, PA, USA)</td><td>525-0150</td><td></td></tr><tr><td>SuperClear centrifuge tubes (50 mL)</td><td>VWR International (Radnor, PA, USA)</td><td>525-0155</td><td></td></tr><tr><td>Thermomixer comfort 1.5 mL</td><td>Eppendorf (Hamburg, Tyskland)</td><td>53,55,27,831</td><td>Temperature controlled mixer</td></tr><tr><td>Trizma base (reagent grade, &amp;ge; 99.0 %)</td><td>Sigma Aldrich (St. Louis, MO, USA)</td><td>T6066</td><td>Tris(hydroxymethyl)aminometan (tris)&amp;nbsp;</td></tr><tr><td>Trizma HCl (reagent grade, &amp;ge; 99.0%)</td><td>Sigma Aldrich (St. Louis, MO, USA)</td><td>T3253-100G</td><td>Tris(hydroxymethyl)aminometan HCl (tris HCl)</td></tr><tr><td>Trypsin (TCPK-treated from bovine pancreas, 10,000-15,000 BAEE units/mg Protein)</td><td>Sigma Aldrich (St. Louis, MO, USA)</td><td>T8802</td><td>Store in freezer below -20 &amp;deg;C</td></tr><tr><td>Tween 20</td><td>Sigma Aldrich (St. Louis, MO, USA)</td><td>P7949-500ML</td><td>polysorbate 20</td></tr><tr><td>Tweezers</td><td>Sigma Aldrich (St. Louis, MO, USA)</td><td>TEM-78511-27</td><td></td></tr><tr><td>Vial caps, white, 9 mm</td><td>Nerliens Meszansky (Oslo, Norge)</td><td>LPP 09 15 0981</td><td></td></tr><tr><td>Mitra microsampler with VAMS (Volumetric adsorptive microsampling) technology, 10 &amp;micro;L, 4-sampler clamshell</td><td>Neoteryx (Torrance, CA, USA)</td><td></td><td></td></tr></tbody></table>

microsampling in targeted mass spectrometry-based protein analysis of low-abundance proteins

This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed using bead-based proteolysis prior to proteotypic peptide affinity-capture and liquid chromatography tandem mass spectrometry (LC-MS/MS) determination. The procedure can be applied to both conventional dried samples using paper cards (e.g., dried blood spots [DBSs] and dried serum spots [DSSs]), as well as samples collected with newer sampling methods such as volumetric absorptive microsampling (VAMS). In addition to describing this procedure, the preparation of both trypsin beads and antibody-coated beads is presented in a step-by-step manner in this work. The advantages of the presented procedure are time-efficient proteolysis using beads and selective robust cleanup using peptide affinity-capture. The current procedure describes the determination of the low-abundance small-cell lung cancer (SCLC) biomarker, progastrin-releasing peptide (ProGRP), in dried serum (both DSSs and VAMS). Detailed procedures for bead preparation make it easier to implement the workflow in new applications or other laboratories. It is demonstrated that the results may be dependent on the sampling material; for the present project, higher signal intensities were seen for samples collected using VAMS compared to DSSs.

An overview of the analytical workflow using both DSS sampling and VAMS is shown in Figure 1. Except for the differences in sampling method, the procedures are identical. Images of the serum sampled using the two sampling methods can be seen in Figure 2.
Both sampling forms (VAMS and DSS) are suitable for the sampling of ProGRP-containing serum. This can be seen from Figure 3 where MS chromatograms of the proteotypic peptide and the IS SIL peptide from DSS and VAMS sampling are shown. In addition, the MS chromatogram after the analysis of a control sample consisting of 10 &#181;L of spiked liquid serum sample processed in the same manner as the dried samples is included. The latter was diluted in the same volume as the volume of the DSS/VAMS extraction solution, subjected to digestion using trypsin beads and cleaned up using peptide affinity capture.
Comparing VAMS and DSS sampling (Figure 4), VAMS provides a higher proteotypic peptide/IS area ratio than DSS. This indicates that there might be a loss of the target protein ProGRP to the paper used for DSS (pure cellulose). When comparing to a control sample, where the serum is not dried prior to further processing and analysis (Figure 4), it is shown that VAMS provides similar area ratios as the control sample (two-tailed t-test, p&#8804; 0.65), indicating no loss to the sampling material, while DSS provides a significantly lower area ratio (two-tailed t-test, p&#8804; 0.005), indicating loss to the sampling material.
A brief evaluation was performed using VAMS. Linearity was shown from 10 to 1,000 ng/mL (R2 = 0.9996), with a limit of detection (LOD, S/N = 3) of 6.7 ng/mL. The LOD is considered satisfactory as the analysis was performed on a rather old triple quadrupole (2008) with a 1 mm ID column. The repeatability of all levels with an S/N &#62; 10 was also considered satisfactory with an RSD between 7% and 17% (n = 3), using IS correction.
Affinity capture can be performed both before and after the digestion step by either capturing the protein of interest or its proteotypic peptide. The current procedure describes peptide affinity capture. An advantage of this approach compared to protein capture is that only the peptide of interest is captured, and an even more efficient sample cleanup is achieved. This is illustrated in Figure 5, showing a more complex full scan chromatogram with more noise after protein capture compared after peptide capture. The samples analyzed in Figure 5 are not sampled using DSS sampling or VAMS; however, serum is also the sample matrix, and affinity capture is performed using the same antibody used for peptide capture in the described procedure.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64473/64473fig01.jpg" /> 
Figure 1: Overview of the analytical workflow using both DSS and VAMS sampling. Abbreviations: DSS = dried serum spot; VAMS = volumetric absorptive microsampling; LC-MS/MS = liquid chromatography-tandem mass spectrometry. <a href="https://www.jove.com/files/ftp_upload/64473/64473fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64473/64473fig02.jpg" /> 
Figure 2: Images of serum sampled using different methods. (A) DBS cellulose card and (B) VAMS. Abbreviations: DBS = dried blood spot; VAMS = volumetric absorptive microsampling. <a href="https://www.jove.com/files/ftp_upload/64473/64473fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64473/64473fig03.jpg" /> 
Figure 3: Representative MS chromatograms of the proteotypic peptide and the IS SIL peptide after DSS and VAMS sampling, as well as for a spiked serum sample added directly into the extraction solution. The MS chromatograms shows 10 &#181;L serum samples spiked with 1.5 &#181;g/mL of ProGRP and applied to (A) VAMS, (B) the cellulose sampling card (for DSS), or (C) directly to the extraction buffer (control sample). Twenty-five microliters of 14 ng/mL IS SIL peptide added to all samples prior to peptide affinity capture. Abbreviations: DSS = dried serum spot; VAMS = volumetric absorptive microsampling; MS = mass spectrometry; ProGRP = progastrin-releasing peptide; IS = internal standard; SIL = stable isotope-labeled. <a href="https://www.jove.com/files/ftp_upload/64473/64473fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64473/64473fig04.jpg" /> 
Figure 4: Representative results of the ALGNQQPSWDSEDSSNFK/IS area ratio for serum samples spiked with ProGRP and applied (10 &#181;L) to VAMS, DSS, or directly to extraction solution (control sample). Concentration of ProGRP is 1.5 &#181;g/mL, n = 4 for each condition; 25 &#181;L of 14 ng/mL IS SIL peptide is added to all samples prior to peptide affinity capture. *indicates that the area ratio is significantly different from samples applied to VAMS (two-tailed t-test, p&#8804; 0.005). Error bars are &#177; standard deviation. Abbreviations: DSS = dried serum spot; VAMS = volumetric absorptive microsampling; ProGRP = progastrin-releasing peptide; IS = internal standard; SIL = stable isotope-labeled. <a href="https://www.jove.com/files/ftp_upload/64473/64473fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64473/64473fig05.jpg" /> 
Figure 5: Comparison of base peak chromatograms (full scan Orbitrap analysis) after intact protein extraction (blue) and proteotypic epitope peptide extraction (red). Extracted ion chromatograms of the proteotypic epitope peptide (ALGNQQPSWDSEDSSNFK, m/z 1005.45) are shown on the right. Serum spiked with 150 ng mL&#8722;1 ProGRP was used as the sample. This figure is reprinted from Levern&#230;s et al.14. <a href="https://www.jove.com/files/ftp_upload/64473/64473fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Table S1: An overview of the composition of buffers and solutions and how to prepare them. <a href="https://www.jove.com/files/ftp_upload/64473/64473_Supp Table S1 Buffers and solutions.xlsx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Microsampling in Targeted Mass Spectrometry-Based Protein Analysis of Low-Abundance Proteins at JoVE.com

Microsampling in Targeted Mass Spectrometry-Based Protein Analysis of Low-Abundance Proteins

A protocol is presented for the determination of low-abundance biomarkers from dried serum samples exemplified with the biomarker progastrin-releasing peptide (ProGRP). Antibody-coated magnetic beads are used for the selective cleanup and enrichment of a proteotypic ProGRP peptide. The captured peptide is subsequently analyzed by liquid chromatography-tandem mass spectrometry.

This paper presents a protocol with detailed descriptions for efficient sample cleanup of low-abundance proteins from dried samples. This is performed ...

microsampling-targeted-mass-spectrometry-based-protein-analysis-low

Research

JoVE Journal

Chemistry

2.3K Views.  University of Oslo. A protocol is presented for the determination of low-abundance biomarkers from dried serum samples exemplified with the biomarker progastrin-releasing peptide (ProGRP). Antibody-coated magnetic beads are used for the selective cleanup and enrichment of a proteotypic ProGRP peptide. The captured peptide is subsequently analyzed by liquid chromatography-tandem mass spectrometry.

Video: Microsampling in Targeted Mass Spectrometry-Based Protein Analysis of Low-Abundance Proteins

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually determined by transcriptomic, proteomic, metabolomics and lipidomic measurements. In this protocol, we introduce a simple method for the reproducible extraction of metabolites, lipids and proteins from biological tissues using a single aliquot per sample. The extraction method is based on a methyl tert-butyl ether: methanol: water system for liquid: liquid partitioning of hydrophobic and polar metabolites into two immiscible phases along with the precipitation of proteins and other macromolecules as a solid pellet. This method, therefore, provides three different fractions of specific molecular composition, which are fully compatible with common high throughput 'omics' technologies such as liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometers. Even though the method was initially developed for the analysis of different plant tissue samples, it has proved to be fully compatible for the extraction and analysis of biological samples from systems as diverse as algae, insects, and mammalian tissues and cell cultures.

A protocol for comprehensive extraction of lipids, metabolites and proteins from biological tissues using one sample is presented.

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

Biochemistry

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Bioengineering

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 &#181;g of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

Here, we describe a chromatographic assay coupled with the ion mobility separation of peptide precursors followed by the high-resolution (~30,000) MS-detection of peptide fragments for the quantification of spiked peptide standards in a monoclonal antibody digest.

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Carrier-assisted One-pot Sample Preparation for Targeted Proteomics Analysis of Small Numbers of Human Cells

Protein analysis of small numbers of human cells is primarily achieved by targeted proteomics with antibody-based immunoassays, which have inherent limitations (e.g., low multiplex and unavailability of antibodies for new proteins). Mass spectrometry (MS)-based targeted proteomics has emerged as an alternative because it is antibody-free, high multiplex, and has high specificity and quantitation accuracy. Recent advances in MS instrumentation make MS-based targeted proteomics possible for multiplexed quantification of highly abundant proteins in single cells. However, there is a technical challenge for effective processing of single cells with minimal sample loss for MS analysis. To address this issue, we have recently developed a convenient protein carrier-assisted one-pot sample preparation coupled with liquid chromatography (LC) - selected reaction monitoring (SRM) termed cLC-SRM for targeted proteomics analysis of small numbers of human cells. This method capitalizes on using the combined excessive exogenous protein as a carrier and low-volume one-pot processing to greatly reduce surface adsorption losses as well as high-specificity LC-SRM to effectively address the increased dynamic concentration range due to the addition of exogeneous carrier protein. Its utility has been demonstrated by accurate quantification of most moderately abundant proteins in small numbers of cells (e.g., 10-100 cells) and highly abundant proteins in single cells. The easy-to-implement features and no need for specific devices make this method readily accessible to most proteomics laboratories. Herein we have provided a detailed protocol for cLC-SRM analysis of small numbers of human cells including cell sorting, cell lysis and digestion, LC-SRM analysis, and data analysis. Further improvements in detection sensitivity and sample throughput are needed towards targeted single-cell proteomics analysis. We anticipate that cLC-SRM will be broadly applied to biomedical research and systems biology with the potential of facilitating precision medicine.

A protein carrier-assisted one-pot sample preparation coupled with liquid chromatography (LC) - selected reaction monitoring (SRM) termed cLC-SRM is a convenient method for multiplexed targeted proteomics analysis of small numbers of cells, including single cells. It capitalizes on using excessive exogenous protein as a carrier and high-specificity LC-SRM for targeted quantification.

Protein analysis of small numbers of human cells is primarily achieved by targeted proteomics with antibody-based immunoassays, which have inherent ...

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

Biology

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, including RNA processing, signal transduction, DNA damage response, miRNA biogenesis, and translation.
In recent years, thanks to biochemical and analytical developments, mass spectrometry (MS)-based proteomics has emerged as the most effective strategy to characterize the cellular methyl-proteome with single-site resolution. However, identifying and profiling in vivo protein R-methylation by MS remains challenging and error-prone, mainly due to the substoichiometric nature of this modification and the presence of various amino acid substitutions and chemical methyl-esterification of acidic residues that are isobaric to methylation. Thus, enrichment methods to enhance the identification of R-methyl-peptides and orthogonal validation strategies to reduce False Discovery Rates (FDR) in methyl-proteomics studies are required.
Here, a protocol specifically designed for high-confidence R-methyl-peptides identification and quantitation from cellular samples is described, which couples metabolic labeling of cells with heavy isotope-encoded Methionine (hmSILAC) and dual protease in-solution digestion of whole cell extract, followed by off-line High-pH Reversed Phase (HpH-RP) chromatography fractionation and affinity enrichment of R-methyl-peptides using anti-pan-R-methyl antibodies. Upon high-resolution MS analysis, raw data are first processed with the MaxQuant software package and the results are then analyzed by hmSEEKER, a software designed for the in-depth search of MS peak pairs corresponding to light and heavy methyl-peptide within the MaxQuant output files.

Protein Arginine (R)-methylation is a wide-spread post-translational modification regulating multiple biological pathways. Mass spectrometry is the best technology to globally profile the R-methyl-proteome, when coupled to biochemical approaches for modified peptide enrichment. The workflow designed for the high confidence identification of global R-methylation in human cells is described here.

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, ...

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous proteomes, including those from viruses, bacteria, and yeast.&#160; In comparison, analysis of the human proteome lags behind, partially due to the sheer number of proteins which must be studied, but also the complexity of networks and interactions these present. To specifically address the challenges of understanding the human proteome, we have developed HaloTag technology for protein isolation, particularly strong for isolation of multiprotein complexes and allowing more efficient capture of weak or transient interactions and/or proteins in low abundance. &#160;HaloTag is a genetically encoded protein fusion tag, designed for covalent, specific, and rapid immobilization or labelling of proteins with various ligands. Leveraging these properties, numerous applications for mammalian cells were developed to characterize protein function and here we present methodologies including: protein pull-downs used for discovery of novel interactions or functional assays, and cellular localization. We find significant advantages in the speed, specificity, and covalent capture of fusion proteins to surfaces for proteomic analysis as compared to other traditional non-covalent approaches. We demonstrate these and the broad utility of the technology using two important epigenetic proteins as examples, the human bromodomain protein BRD4, and histone deacetylase HDAC1.&#160; These examples demonstrate the power of this technology in enabling &#160;the discovery of novel interactions and characterizing cellular localization in eukaryotes, which will together further understanding of human functional proteomics. &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;

HaloTag technology is a multifunctional technology which has shown significant success in isolation of both small and large protein complexes from mammalian cells.&#160; Here we highlight the advantages of this technology compared to existing alternatives and demonstrate its utility to study numerous aspects of protein function inside eukaryotic cells.

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...