WO2015077660A1 - Enzyme reactor system enabling enhanced enzymatic digestion, analyte solubility and improved mass spectrometric compatibility - Google Patents

Abstract

Methods and systems for protein hydrolysis with optimized buffer composition compatible to Mass spectrometry are disclosed.

Description

ENZYME REACTOR SYSTEM ENABLING ENHANCED ENZYMATIC DIGESTION, ANALYTE SOLUBILITY AND MASS SPECTROMETRY COMPATIBILITY

CROSS REFERENCE

This application claims the priority of US provisional applications US 61/908,214, filed on Nov. 25, 2013; US 62/025,783, filed on July 17, 2014; and US 62/069,780, filed on Oct. 28 2014. The application is also a continuation in part for PCT/US14/13782, published as WO 2014/120890 on August 7, 2014. The disclosures therein are incorporated into this application entirely.

Field of Invention

This invention discloses an innovative enzyme reactor working system. Specifically, the enzyme reactor working system has buffer compositions, an enzyme reactor, digestion methods and reactor formats for enhanced enzymatic digestion, analyte solubility and mass spectrometric compatibility. The system provides improved digestion, recovery and parallel processing capabilities for both qualitative and quantitative processing of hundreds of difficult to digest samples simultaneously.

Background

The growing impact of proteins as efficacious drugs and diagnostic biomarkers is forcing the analytical community to deal with extremely high levels of analyte and sample complexity. However, due to the time and cost associated with developing an enzyme linked immunoassay (ELISA), mass spectrometric approaches are playing an increasing role in protein analyses. Furthermore, mass spectrometric methods enable detection of isoform variations and post- translational modifications; identification of these features being key to an understanding of protein function and activity.

Applications of enzymatic catalyses in biotechnology are limited by the activity, stability and specificity of the enzyme under desired operating conditions. As such, the discovery and/or development of enzyme working systems with improved characteristics can be the impetus for scientific breakthrough. Notably, Thermus aquaticus (Taq) polymerase has become one of the most important enzymes in molecular biology due to its ability to withstand the protein- denaturing conditions (high temperature) required during polymerase chain reaction (PCR) temperature cycling. While many historical attempts to apply such enzymes to proteomics have resulted in restricted success, an increasingly large number of applications are being developed using technologies that enable simultaneous denaturation and digestion (PCT/US14/13782). Digestion under elevated temperature conditions results in a significant time savings and often dramatic simplification of the workflow. However, the use of elevated temperature often leads to protein denaturation and, as a consequence, protein precipitation. Precipitation negatively impacts the workflow by slowing the reaction while also making the sample incompatible with traditional LC/MS analyses. Therefore, it is a prerequisite that elevated temperature reactions be paired with a strategy for effectively maintaining substrate solubility. Furthermore, the ideal enzyme reactor system would be composed of materials that are either compatible with mass spectrometric analyses or otherwise easily removed.

Therefore, an enzyme working system that would enable enhanced hydrolysis at elevated temperatures, and sustained substrate solubility composed of materials either compatible with mass spectrometric analyses or otherwise easily removed is desired.

Brief description of Figures

Figure 1 : Peptide Formation as a Function of Digestion Temperature (peptide m/z = 938)

Figure 2: Peptide Formation as a Function of Digestion Temperature (peptide m/z = 603)

Figure 3: Peptide Formation as a Function of Digestion Buffer (solution digestion)

Figure 4: Peptide Formation as a Function of Digestion Buffer (IMER digestion)

Figure 5: 50 mM ammonium bicarbonate, 1M CaC12 - Precipitate Formed during Heating

Figure 6: 50 mM ammonium bicarbonate, 5mM CaC12 - Precipitate Formed during Heating

Figure 7: The Effect of CaC12 Concentration on Digestion Using Ammonium Acetate Buffer in an Immobilized Enzyme Reactor

Figure 8: Peptide Formation as a Function of Digestion Buffer Part 2 (IMER digestion)

Figure 9: The Effect of Buffer Composition on Digestion in an IMER at Various Temperatures Summary of the Invention

This disclosure provides an enzyme working system that would enable enhanced hydrolysis at elevated temperatures and sustained substrate solubility comprising materials either compatible with mass spectrometric analyses or otherwise easily removed.

In some preferred embodiments the enzyme working system comprises essentially the following components: a. an enzyme reactor that provides rapid hydrolysis to at least one substrate, wherein the hydrolysis utilizes an enzyme selected from the group consisting of trypsin, chymotrypsin, Lys-C, Glu-C, Arg-C, Asp N, papain, pepsin, elastase, IdeS, pronase and PNGase F and a combination thereof; and

b. a reaction buffer comprising sugar alcohols or sugars selected from the group consisting of glycerol, glycol, trehalose, glucose, fructose, sucrose, maltose, lactose, galactose, allose, altrose, mannose, gulose, idose, talose, sorbitol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol and inositol.

In some preferred embodiments, the enzyme working system is further comprised of a metal cation concentration greater than 1 nanomolar in said reaction buffer, wherein the metal cation in the reaction buffer is selected from the group consisting of aluminum, calcium, copper, iron, magnesium, manganese, mercury, sodium and silver.

In some preferred embodiments, said hydrolysis is conducted by an enzyme in solution, an immobilized enzyme reactor, microwave reactor, ultrasound, infrared, filter aided sample preparation, solvent aided trypsin digestion, a pressure reactor or any combination thereof.

This disclosure also provides an enzyme working system comprised of an immobilized enzyme and an enzyme reaction buffer that is compatible with mass spectrometric analyses. The system enables enhanced enzymatic digestion and prevents protein aggregation at elevated temperatures, and the reaction buffer of the system comprises an acetate salt in combination with metal concentration greater than 1 nanomolar.

In some preferred embodiments of the aforementioned enzyme working system: a. the immobilized enzyme that provides rapid hydrolysis to at least one substrate is selected from the group consisting of trypsin, chymotrypsin, Lys-C, Glu-C, Arg-C, Asp N, papain, pepsin, elastase, IdeS, pronase and PNGase F or a combination thereof; b. the reaction buffer comprising acetate salts is selected from the group consisting of aluminum acetate, ammonium acetate, potassium acetate, propyl acetate, phenyl acetate, octyl acetate, dodecyl acetate, geranyl acetate, glycerin acetate, amyl acetate, vinyl acetate, methyl acetate, ethyl acetate, isopropyl acetate, ethylhexyl acetate, butyl acetate, sodium acetate, copper acetate, calcium magnesium acetate, cesium acetate, barium acetate, beryllium acetate, cadmium acetate, magnesium acetate, chromium acetate, iron acetate, lead acetate, manganese acetate, sodium diacetate, lithium acetate, magnesium acetate, mercury acetate, molybdenum acetate, nickel acetate, palladium acetate, platinum acetate, rhodium acetate, silver acetate, triethylammonium acetate, zinc acetate.

c. the metal cation in the reaction buffer is selected from the group consisting of aluminum, calcium, copper, iron, magnesium, manganese, mercury, sodium and silver.

This disclosure also provides an enzyme working system comprised of an immobilized enzyme and an enzyme reaction buffer that is compatible with mass spectrometric analyses. The system enables enhanced enzymatic digestion and prevents protein aggregation at elevated temperatures, and the reaction buffer of the system comprises a volatile salt in combination with metal concentration greater than 1 nanomolar.

In some preferred embodiments of the aforementioned enzyme working system: a. the immobilized enzyme that provides rapid hydrolysis to at least one substrate is selected from the group consisting of trypsin, chymotrypsin, Lys-C, Glu-C, Arg-C, Asp N, papain, pepsin, elastase, IdeS, pronase and PNGase F or a combination thereof;

b. the reaction buffer comprising volatile salts is selected from the group consisting of ammonium acetate, ammonium formate, pyridine acetate, N-ethylmorpholine acetate, trimethylammonium acetate c. the metal cation in the reaction buffer is selected from the group consisting of aluminum, calcium, copper, iron, magnesium, manganese, mercury, sodium and silver.

In some preferred embodiments, the aforementioned enzyme working systems further comprise either a surfactant, or detergent selected from the group consisting of octylglucoside, sodium dodecyl sulfate, sodium laureth sulfate, ammonium lauryl sulfate, sodium pareth sulfate, Big CHAP deoxy, ASB, sodium deoxycholate, an acid-cleavable detergent or a combination thereof.

In some preferred embodiments, the aforementioned working systems further comprise either a sugar alcohol or sugar selected from the group consisting of glycerol, glycol, trehalose, glucose, fructose, sucrose, maltose, lactose, galactose, allose, altrose, mannose, gulose, idose, talose, sorbitol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol and inositol or a combination thereof.

In some preferred embodiments, the aforementioned enzyme working systems are operated at elevated temperature of 37°C or above.

In some preferred embodiments, the enzyme is a modified enzyme selected from the group consisting of modified trypsin, chymotrypsin, Lys-C, Glu-C, Arg-C, Asp N, papain, pepsin, elastase, IdeS, pronase and PNGase F or a combination thereof.

In some preferred embodiments, the immobilized enzyme reactor conduct rapid hydrolysis in the format of microwave reactor, ultrasound, infrared, filter aided sample preparation, solvent aided trypsin digestion, a pressure reactor or any combination thereof.

In some preferred embodiments, the reaction buffer formulations further comprise co- solvents including but not limited to organic solvents, chaotropes, surfactants, detergents, salts, sugars, sugar alcohols, and any combination thereof.

In some preferred embodiments the substrate of the enzyme is not denatured, reduced, alkylated either before or after hydrolysis.

In some preferred embodiments the substrate of the enzyme is denatured, reduced, alkylated either before or after hydrolysis. In some preferred embodiments the reactor format is a combination heater/shaker instrument, a heating block on a shaker, shaking in a convection oven, shaking in a water bath, shaking in an incubator, shaking in a microwave oven or any combination thereof.

In some preferred embodiments the reactor comprising a reactor vessel in the form of a thin walled PCR tube, any thin walled sample tube, or multi-well plate.

In some preferred embodiments the reactor is in the form of a column, eppendorf tube, pipette tip, multi well plate, or magnetic particle.

In some preferred embodiments the immobilized enzyme reactor comprising supporting material that is selected from the group consisting of polystyrene, polystyrene/divinylbenzene, silica, controlled porosity glass, dextrans, agarose, acrylates, magnetic particles and nitrocellulose.

In some preferred embodiments the immobilized enzyme reactor supporting material is in a form of particle, monolithic, membrane, planar or microfluidic channel.

These and other features, aspects and advantages of the present invention will become better understood with reference to the following figures, associated descriptions and claims.

Detailed Description

This disclosure provides an enzyme working system that would enable enhanced hydrolysis at elevated temperatures and sustained substrate solubility composed of materials either compatible with mass spectrometric analyses or otherwise easily removed.

While many historical attempts to apply temperature stable enzymes to proteomics have resulted in restricted success, an increasingly large number of applications are being developed using technologies that employ temperature stable enzymes thereby enabling simultaneous denaturation and digestion (PCT/US14/13782). Digestion under elevated temperature conditions often results in a significant time savings, increase in peptide signal and dramatic simplification of the workflow (see Example 3 below). However, the use of elevated temperature often leads to protein denaturation and as a consequence protein precipitation (see Example 4 below). Precipitation negatively impacts the workflow by slowing the reaction while also making the sample incompatible with traditional LC/MS analyses. Therefore, it is a prerequisite that elevated temperature reactions be paired with a strategy for effectively maintaining substrate solubility. One precipitation mitigation strategy is to apply rapid digestion at elevated temperatures (e.g. Example 3). Example 5 shows that in general precipitation is mitigated as a result of digestion. However, additional improvement was needed in order for this strategy to be applied to complex matrices such as plasma.

It was determined that the use of a tris buffer and high concentrations of CaC12 in combination with glycerol enables the digestion of 4x diluted plasma at elevated temperatures (e.g. Examples 8, 12-14). While tris buffer and high concentrations of CaC12 can negatively impact mass spectrometric sensitivity by means of ion suppression, they are easily removed by means of desalting or solid phase extraction. However, in some cases it is desirable to avoid such desalting and solid phase extractions steps. As such, attempts were made to develop an enzyme reactor system with improved mass spectrometric compatibility in addition to enhanced enzymatic digestion and analyte solubility.

Ammonium bicarbonate is a volatile salt, thermally decomposing to non-ionic products. As such, the use of ammonium bicarbonate has minimal impact on ionization. It is because of this phenomenon that ammonium bicarbonate is frequently used as a digestion buffer (Promega Technical Manual # 9PIV511 ; Becker and Hoofnagle, 2012; Blonder et al., 2006; Fusaro et al., 2009; Kettenbach et al., 2011; Pramanik et al., 2002; Shevchenko et al., 2006; Wisniewski et al., 2009) prior to LC-MS/MS analyses.

Comparisons of digestion buffers used when performing tradition solution based digestion protocols showed that enzyme activity associated with the use of ammonium bicarbonate is significantly higher than enzyme activity associated with the use of tris buffer containing calcium chloride (see Example 15 below). In contrast, comparisons of digestion buffers used when performing digestions in an enzyme reactor showed that enzyme activity associated with the use of tris buffer containing calcium chloride is significantly higher than enzyme activity associated with the use of ammonium bicarbonate (see Example 16 below). Attempts to improve the performance of ammonium bicarbonate in an enzyme reactor by means of adding calcium chloride to the ammonium bicarbonate resulted in a precipitate.

Serendipitously, it was found that a combination of ammonium acetate and calcium chloride provided improved enzyme activity (see Example 17 below). This combination also enables direct injection of the digested samples into an LC/MS/MS instrument with minimal ion suppression. Furthermore, the improvement in enzyme activity over ammonium bicarbonate was found to be increasingly dramatic as digestion temperature increases (see Example 19 below). As such, combinations of ammonium acetate and calcium chloride enable the digestion of proteins under denaturing, elevated temperature conditions. It was also determined that additions of detergents as well as a sugar alcohols could improve protein solubility during digestion under denaturing conditions (see Examples 21-23 below).

An important, non-obvious, observation was that the effects of calcium chloride on the digestion buffer are buffer dependent. The performance of tris as a digestion buffer was dramatically improved by means of using unusually high concentrations of calcium chloride (Examples 11). The performance of ammonium acetate as a digestion buffer was dramatically improved by means of using low concentrations of calcium chloride, while increasing concentrations of calcium chloride had negative effects on the performance of ammonium acetate as a digestion buffer (e.g. Example 17).

Enzymes

An exemplary proteolytic enzyme for use in digestion is trypsin. While the most popular proteolytic enzyme is trypsin, other enzymes with alternative functionalities may also be employed, such as chymotrypsin, Lys-C, Glu-C, Arg-C, Asp N, papain, pepsin, elastase, IdeS, pronase and PNGase F or combinations thereof, so that a variety of peptide products can be generated increasing the protein sequences that are observed and sequenced to provide more definitive identifications.

Formats

An exemplary format for digestion at elevated temperatures is a temperature stable immobilized enzyme. However, soluble trypsin, a microwave reactor, ultrasound, infrared, filter aided sample preparation, solvent aided trypsin digestion, a pressure reactor or any combination thereof may also be employed.

Stationary Phase Materials

An exemplary solid-support material for use in an immobilized enzyme reactor (IMER) is polystyrene/divinylbenzene (PS/DVB) material. Other suitable solid-support materials may include other polystyrene-based materials, silica-based materials, controlled porosity glass and nitrocellulose-based materials, as well as magnetic materials. The solid-support material may be in a particle form, or the solid-support material may be monolithic in form, a membrane, or planar in form. Also, microfluidic channels and the like may be advantageous.

Magnetic bead format

It may also be the case where that enzyme is directly immobilized on a magnetized particle. In this case the material would reside in either a tube or plate format. During incubation the tube or plate can be loaded into a heater or combination heater/shaker apparatus. After incubation the plate assembly is brought into proximity with a magnet so as to enable the separation of the immobilized enzyme from the analyte solution.

Column Format

One form of the immobilized enzyme involves slurry packing into an open tubular/column system. The column can then be end-capped with liquid permeable frits. These frits provide a selective barrier, enabling the passage of sample solutions and precluding the passage of the immobilized enzyme. As such, samples and solutions can be pumped through the column by means of liquid chromatography (LC) pumping systems. The column housing itself should provide sufficient heat conduction. This column format is well suited for automation.

Multi-well Formats

During sample preparation it is often desirable to process multiple samples at once, in parallel. Furthermore, such formats may be disposable such that the possibility of cross- contamination is limited. Such parallel processing enables significant time savings and can promote uniformity. In such cases an enzyme reactor in the form of a multi-well or plate format is ideal. In such cases, uniform heat conduction across the multiple wells is important.

Use of a thermo cycler

Analytical reproducibility depends on reproducible heating of the enzyme during the sample preparation period. It is important that the heating apparatus is capable of providing uniform heating to all samples. One example as illustrated in this disclosure is a PCR thermo cycler. It is particularly well suited for this operation as its heated surface is usually made from a material that enables rapid heat transfer (such as silver), the heated sample interface extends a significant distance up the sides of the tube, and heat is provided from the top of the apparatus to eliminate condensation and further promote uniform results. While a thermo cycler has proven efficacious, it is possible that alternative heating apparatuses such as heating blocks, multichannel heaters, water baths, ovens, microwave ovens or combinations thereof are also capable of providing the requisite uniform heating.

Use of a combination heater/shaker

At any given time enzyme reactions are limited by either a lack of enzyme or diffusion limitations. In the column format, diffusion limitations can be minimized by pumping the mobile phase through materials containing very large pores. In these situations the stationary phase behaves similarly to a monolith. In the multi-well formats, diffusion limitations can be minimized through the use of agitation. In these cases sufficient mixing increases the reaction rate. An exemplary approach is to put the enzyme into an apparatus that is undergoing vigorous shaking/heating. Alternatively, agitation in the form of rotation, end over end mixing, the use of a stir bar, sonicating (ultrasound), repeat pipetting or combinations thereof could be capable of providing the requisite agitation while a combination heater/shaker instrument, shaking in a convection oven, shaking in a water bath, and shaking in a microwave oven are example combinations capable of providing both heating and mixing.

Detectors

It is also within the scope of the present disclosure that the system may include a UV absorbance or fluorescence monitor for further analysis.

Example 1: Making improved immobilized enzyme

In a 50 mL reaction vessel were added 1.334 mL epibromohydrin, 0.666 mL glycidol, 15 mL dichloromethane and 50 uL boron trifluoride etherate. This reaction was allowed to incubate at room temperature for 24 hrs. After 24 hours the solvent was removed en vacuo. To this same reaction vessel 10 mL dH₂0, 7 mL polyethylene glycol, 30 mL IPA and 3 g PS-DVB were added. This flask was gently agitated for 14 hours. The resulting coated particle were filtered and collected in a clean round bottom flask. 10 mL 2 M KOH was added and the mixture gently agitated for 2 hours. After 2 hours 2 g sorbitol was added and the mixture gently agitated at room temperature for 12 hours. After 12 hours this mixture was filtered, washed with water and transferred to a clean round bottom flask. 100 mg sodium periodate was added to these materials and the resulting mixture incubated for 1 hour at room temperature. After 1 hour these materials were washed with 20 mL 50 mM sodium carbonate, filtered and collected into a large eppendorf.

150 mg of this material was added to a 15 mL eppendorf. To a separate eppendorf was added 1.2 g sodium sulfate diluted to 10 mL with water. 30 mg sodium cyanoborohydride was added to a separate 1.5 mL eppendorf and diluted with 1.5 mL 100 mM carbonate buffer, pH 9.6. 30 mg benzamidine was added to a separate 1.5 mL eppendorf and diluted with 1.5 mL 100 mM carbonate buffer, pH 9.6. 30 mg porcine trypsin was added to a separate 1.5 mL eppendorf and diluted with 600 uL of the benzamidine solution and transferred to the eppendorf containing the resin. To this resin containing eppendorf 720 uL of the sodium sulfate solution was added, 81 uL of the sodium cyanoborohydride solution was added and 840 uL of the 100 mM carbonate was added. This reaction mixture was gently agitated for 18 hours at room temperature. After 18 hours 200 mg benzamidine was added to a 15 mL eppendorf. The benzamidine was diluted in 10 mL carbonate buffer. The reaction mixture was centrifuged at 2000 rpm for 60 seconds, the solution was decanted, 2.5 milliliters of the fresh benzamidine solution added and the mixture vortexed. This wash cycle was repeated 2 more times so any unreacted, excess trypsin was removed. 30 mg sodium cyanoborohydride was added to a separate 1.5 mL eppendorf and diluted with 1.5 mL 100 mM carbonate buffer, pH 9.6. 81 uL of this solution was added and the reaction mixture gently agitated at room temperature for 4 hours. After 18 hours 200 mg benzamidine was added to a 15 milliliter eppendorf. 10 mL carbonate buffer was added to the benzamidine. The reaction mixture was centrifuged at 2000 rpm for 60 seconds, the solution decanted, 2.5 mL of the fresh benzamidine solution added and the mixture vortexed. This wash cycle was repeated 2 more times. 8 mg of acetic acid N-hydroxy succinimide was then added to the resin and the resulting reaction mixture incubated for 2 hours at room temperature. After 2 hours the resulting product was washed once with 100 mM carbonate buffer and 2 additional times with DMSO. 15 mg TPCK was added to a clean 1.5mL eppendorf and dissolved in 200 mL DMSO. This solution was added to the resin and allowed to react for 2 hours. The resulting product was washed once with DMSO then 2 more times with tris-buffer saline pH 7.4.

Nitrogen analysis of these materials showed nitrogen content of 0.26%. This equates to a nitrogen content of 0.0026g nitrogen/gram resin or 2.6mg nitrogen/g resin or roughly 2.6mg/mL. Using the Kjeldahl Method (2013. Critical Reviews in Analytical Chemistry 43: 178-223) one can assume protein is roughly 16% nitrogen content. Therefore the 2.6 mg/mL is multiplied by 6.25 (equal to 100/16). As such one can consider the protein load of the final materials to have been approximately 16 mg trypsin/g resin.

Example 2: Immobilized enzyme PCR strip of 8 format

15uL of the immobilized enzyme prepared according to Example 1 was pipetted into each well of a PCR strip of 8.

Example 3: Digestion efficiency as a function of elevated temperature

Briefly, temperature stable immobilize enzyme was synthesized according to Example 1 and packed in a strip of 8 format prepared according to Example 2. Samples were added directly to this slurry without pretreatment (no upfront denaturation, reduction or alkylation was employed). Samples were heated using an Eppendorf ThermoMixer C equipped with a PCR block and a heated lid set to 70°C, 1400 RPM. Following incubation the entire sample was removed using a pipette and transferred to a clean Eppendorf tube, centrifuged, decanted and the supernatant analyzed by liquid chromatography/mass spectrometry.

Human, monoclonal IgGl was used in this experiment. The digestion efficiency as a function of temperature was evaluated by measuring the peak areas corresponding to the two peptides VVSVLTVLHQDWLNGK (SEQ ID NO 1) and TTPPVLDSDGSFFLYSK (SEQ ID NO 2) as function of digestion time and temperature. These two peptides are unique to human IgG.

Figures 1 and 2 provide a visual representation of the increase in peptide generation as a function of increasing digestion temperature.

Table 1 summarizes the mass spectrometric parameters used for the analysis of the IgGl digested at various times and temperatures using a temperature stable immobilized enzyme reactor

Table 1- The mass spectrometric parameters used for the analysis of the IgGl digested at various times and temperatures using a temperature stable immobilized enzyme reactor

Sample lug/mL human IgG Injection Volume 5 uL

Reversed Phase A 2% ACN (aq) 0.1% Formic Acid

Reversed Phase B 90% ACN (aq) 0.1% Formic Acid

Reversed Phase Gradient 2-70%B in 5 minutes

SEQ ID NO 1 : MS1/MS2 VVSVLTVLHQDWLNGK- 603.82/805.62

SEQ ID N0 2: MS1/MS2 TTPPVLDSDGSFFLYSK - 938.02/805.26

Table 2 shows the peaks areas associated with digestion of human IgG at various digestion times and temperatures using a temperature stable immobilized enzyme reactor

Table 2 The peak areas associated with digestion of human IgG at various digestion times and temperatures using a temperature stable immobilized enzyme reactor

Example 4: Protein aggregation at various concentrations at elevated temperature

The data generated in Example 3 provides empirical evidence of increased digestion efficiency at 70°C in comparison to 60, 50 and 40°C. However, additional problems arise in that, as temperatures rise, substrate denaturation often leads to precipitation. This precipitation negatively changes the reaction kinetics and makes the sample incompatible with traditional LCMS analyses. As an example various concentrations of bovine serum albumin were heated to 70°C in tris buffered saline, lOmM CaCl₂ (serum albumin is the most abundant protein in blood plasma) and then checked periodically for the formation of aggregates. A summary of the results can be seen in Table 3 below.

Table 3 Aggregation of various concentrations of BSA at elevated temperatures

N= No aggregation

Y= Aggregation

S= Slight aggregation

In the case of bovine serum albumin, aggregation is most prevelant at 70°C. However, it is important to note that many proteins aggregate at much lower temperatures.

Examle 5: Aggregation at elevated temperatures in the presence of trypsin In order to determine the effect of trypsin digestion, using soluble trypsin, on solubility various concentrations of bovine serum albumin were heated to 70°C in tris buffered saline, lOmM CaCl₂. Porcine trypsin was added resulting in a 1 :5 trypsin :protein ratio and the samples checked periodically for the formation of aggregates. A summary of the results can be seen in Table 4 below.

Table 4: Aggregation of various concentrations of BSA at elevated temperatures in the presence of trypsin

N= No aggregation

Y= Aggregation

S= Slight aggregation

It was the goal of these experiments to find buffer conditions that would enable the direct digestion of a solution containing at least 5% blood plasma (approximately 3.75 mg/mL). While there were fewer examples of aggregation in the presence of trypsin, the precipication occuring at 3.75 and 6.25 mg/mL protein concentrations suggests that trypsin digestion alone does not sufficiently prevent aggregation.

Example 6: Screening of various additives for the prevention of aggregation

A screen of various additives for the prevention of aggregation was performed using 12.5mg/mL BSA as a model system. The final concentration of the additive is listed in Table below. The starting buffer was comprised of 50mM TBS, lOOmM CaCl₂. Samples comprised of 12.5 mg/mL BSA in buffer/additive mixture were added to temperature stable immobilize enzyme was synthesized according to Example 1 and packed in a strip of 8 format prepared according to Example 2. Finally, the IMER containing the sample was incubated on a ThermoMixer C at 70°C rotating at 1400RPM. Aggregation was monitored as a function of time. A summary of the additive screen can be found in Table 5 below.

Table 5: A screen for the effect of additives on aggregation

N= No aggregation

Y= Aggregation

S= Slight Aggregation

DMF 20% Y Υ Υ Υ

Guanidine HC1 0.5M N Ν Ν Ν

Tween 20 0.005% Y Υ Υ Υ

Tween 20 0.05% Y Υ Υ Υ

Glycerol 5% Ν Ν Ν Ν

Glycerol 10% Ν Ν Ν Ν

Octylglucoside Ν Ν Ν Ν

Deoxycholate Υ Υ Υ Υ

π τ— TFE= trifluoroethanol, 6) DMF= dimet ylformamide

Example 7: Buffer screening- IMER

While the absence of aggregation is a factor to consider when developing an optimal buffer, even in the absence of aggregation it is important that the buffer used not dramatically, negatively impact digestion efficiency. However, a buffer that prevents aggregation but only slightly impacts digestion may be of significant practical value. With these considerations in mind a significant number of buffer conditions were screened using insulin as a model protein. Briefly, digestion was performed using an immobilized enzyme prepared according to Example

1 packed in to a strip of 8 format prepared according to Example 2. A 5mg sample of USP standard human insulin was dissolved in 2% acetic acid, 100 mM glycine to a concentration of 10 mg/mL. This solution was then diluted 100 fold into the digest buffer of interest. The digest buffer varies according to the descriptions in Table 7. 200uL of this sample was added to each of

2 wells. Each sample was digested for 60 seconds at 70°C using a Veriti Thermo Cycler. The sample was then removed from the instrument and allowed to cool for 5 minutes. Once all of the samples had cooled the 2 wells of sample from each buffer condition were pooled, centrifuged and the digest materials decanted. These materials were analyzed by LC-UV/Vis according to parameters in Table 6 below. Table 6: Conditions used for the analysis of insulin samples digested in various buffers

Results are summarized in Table 7. Concentration of buffer and salt had minimal impact while the concentration of CaCl₂ impacted results dramatically. An increase in digestion was observed using concentrations of CaCl₂ as high as 500 mM.

Table 7: Screening effect of buffer composition on digestion in novel immobilized enzyme reactor

(50 m VI)

Peptide Digested Undigested

HEPES

1040553 2438065 1152957 (50 m VI)

CaCl₂ Peptide Digested Undigested

0.05M 1172085 2725600 802494

0.1M 1221898 2844924 609138

0.25M 1264212 2950068 571246

0.5M 1290650 3031625 234332

1M 1263872 3066738 16129

Tris Peptide Digested Undigested

0.05M 1044238 2479332 893529

0.1M 1053652 2540889 990996

0.25M 986402 2440181 1291934

0.5M 879474 2205308 1490447

1M 745060 1923103 2177969

NaCl Peptide Digested Undigested

0.1M 925368 2262987 1557233

0.25M 866812 2119184 2102900

0.5M 862093 2146822 1679944

1M 944511 2363025 1205692

2M 966823 2660004 332026

_PH Peptide Digested Undigested 7 794188 1848671 2145406

7.5 900986 2193062 1498921

8 1236949 3106115 2004796

8.5 979047 2479154 1586443

9 855022 2157753 1785229

Gdn HC1⁴ Peptide Digested Undigested

0.5M 309390 723258 4114993

1M 179523 423606 4328875

2M 56700 136773 4244827

4M 15000 40578 4562051

6M 2447 9402 4396495

Urea Peptide Digested Undigested

0.5M 715848 1816572 2285541

1M 555954 1448998 2812910

2M 349854 905391 3812799

4M 104200 285873 4609299

6M 49088 139668 4675789

8M 13979 43865 4151508

Glycerol Peptide Digested Undigested

5% 828171 2051870 2217803

10% 697488 1779261 2432362

20% 445736 1167862 3174668

OGS⁵ Peptide Digested Undigested 0.05% 958775 2394275 1730676

0.1% 891922 2255277 2114234

0.15% 942341 2430665 2160281

0.2% 891922 2255277 2114234

TWEEN Peptide Digested Undigested

0.01% 931773 2354232 1769078

0.05% 942093 2463078 2182869

0.1% 893558 2346455 1935980

Zwittergent Peptide Digested Undigested

0.1% 175608 501992 5082632

0.2% 103964 316287 4821535

0.5% 53806 185582 5030199

1% 34103 137448 5242668

SDC⁶ Peptide Digested Undigested

0.1% 101770 296346 4216641

0.2% 30902 94589 5010947

0.5% 1351 12088 1648635

1% 1275 8134 284843

DMSO⁷ Peptide Digested Undigested

5% 719810 1804007 2280353

10% 618353 1483343 2811668

15% 437967 1058609 3394971

20% 296540 722730 3631216 ACN⁸ Peptide Digested Undigested

5% 568057 1344796 3327305

10% 309638 719966 4860471

15% 116402 292205 5326017

20% 86754 194140 6433237

MeOH⁹ Peptide Digested Undigested

5% 627591 1511720 2902781

10% 417070 1001203 3946896

15% 277958 675569 5012221

20% 213935 518131 5268985

IPA¹⁰ Peptide Digested Undigested

5% 291902 696091 4427643

10% 86945 208098 5774155

15% 52332 126129 5930073

20% 55155 135325 5148164

Formamide Peptide Digested Undigested

5% 580956 1424720 3061491

10% 305599 745680 3827192

15% 155245 375288 4129486

20% 82549 200621 4430620

TFE¹¹ Peptide Digested Undigested

5% 208344 513982 4502764 10% 39996 95904 5112793

15% 18047 40955 5055881

20% 9756 26014 5626809

1) ABC = ammonium bicarbonate 2) PBS = phosphate buffered saline 3) TBS = tris buffered saline 4) Gdn HC1 = guanidine hydrochloride 5) OGS = octylglucoside 6) SDC = sodium deoxycholate 7) DMSO = dimethylsulfoxide 8) ACN = acetonitrile 9) MeOH = methanol 10) IPA = isopropyl alcohol 11) TFE - tetrafluoroethylene

As Table 7 indicates, digestion efficiency increased dramatically in the presence of high concentrations of calcium chloride. Although it is not clear what makes increased CaCl₂ concentration associate with higher trypsin digestion efficiency, it is clear that the higher concentration of CaCl₂ has a synergistic effect on trypsin digestion. It is contemplated that other metal ions may have a similar effect on enzyme at elevated temperature. For example, Na⁺, Mg²⁺, Ca²⁺, Sr²⁺, Mn²⁺, Co²⁺, Ni²⁺, Zn²⁺, Cu²⁺, Fe³⁺ in various buffer solutions with high ionic strength are expected to show temperature dependent increase in enzyme reaction. One non- limiting explanation is that the metal presence in the reaction buffer, especially in the IMER format enzyme reactions, dramatically increases the chances of enzyme to contact substrate by unknown means. Another non-limiting explanation of the metal effect in the reaction buffer is that the enzyme may be transformed by the presence of metal to become a more active format, such as undergoing conformational change to have altered Kcat/Km ratio that favors higher reaction efficiency. Regardless, it is a surprising discovery from this disclosure that metal concentration can be revised further from the currently available art and protocols to improve the enzyme reaction efficiency at elevated temperatures.

While various literature references describe the use of additives for the prevention of aggregation (Shiraki et al. 2003. Science and Technology of Advanced Materials 4:55-59.; Shiraki et al. 2002. The Journal of Biochemistry 132:591-595.) it is important to note the non- obviousness of these findings. While some additives have precedent as beneficial to the prevention of aggregation they were not beneficial for use in this working system. Also of note, it was observed that heating the sample in the presence of the immobilized enzyme decreased the propensity of the sample to aggregate. While not wishing to be limited to theory, it is possible the digestion of the sample from protein into more soluble peptides increases the overall solubility of the reaction mixture. Example 8: Screening of buffer/additive combinations likely to enable plasma digestion

It is important that the final reaction buffer enable a rapid, complete digestion without experiencing aggregation. As a follow-up to Examples 6 and 7, 150uL each 10% glycerol, 0.1% Octylglucoside and 10%> formamide in 50mM TBS, lOOmM CaCl₂ were added 50uL beagle plasma. These samples were added to an immobilized enzyme made according to Example 1 and packed into a strip of 8 format according to Example 2 then incubated on a ThermoMixer C for 90 minutes. Following incubation, samples using buffers comprised of 0.1 % Octylglucoside and 10% formamide in 50mM TBS, lOOmM CaCl₂ showed significant aggregation. Following incubation the sample comprised of 10% glycerol in 50mM TBS, lOOmM CaCl₂ showed no aggregation. It is contemplated that other sugar alcohols or sugars may have a similar effect on aggregation at elevated temperature. For example glycol, trehalose, glucose, fructose, sucrose, maltose, lactose, galactose, allose, altrose, mannose, gulose, idose, talose, sorbitol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol or inositol are expected to prevent protein aggregation during digestion at elevated temperatures.

Example 9: Buffer screening- Trypsin in solution

While the previous examples were performed using an immobilized enzyme format it came to our attention that these non-obvious effects of CaCl₂ might also improve solution digestion. Using insulin as a model protein a significant number of buffer conditions were screened. Digestion was performed using a commercially available porcine trypsin. Briefly, a 5mg sample of USP standard human insulin was dissolved in 2% acetic acid, 100 mM glycine to a concentration of 10 mg/mL. This solution was then diluted 50 fold, to 200ug/mL, into digest buffer. The digest buffer varies according to the descriptions in Table .Starting with an 80ug/mL solution of trypsin in 20mM acetic acid, trypsin was added to achieve a 1 :10 enzyme: protein ratio. Each sample was digested for 18 hours at 37°C using a Veriti Thermo Cycler. Following digestion, lOOuL of sample was removed and acidified, quenching the reaction. These materials were analyzed by LC-UV/Vis according to parameters in Table 8 below.

Table 8: Conditions used for the analysis of insulin samples digested in various buffers

Sample lOOug/mL digested Hu insulin Injection Volume 25uL

Digest Conditions 70°C, 60 seconds

2% ACN (aq) 0.1%

Mobile Phase A

Trifluoroacetic Acid

90% ACN (aq) 0.1%

Mobile Phase B

Trifluoroacetic Acid

Reversed Phase Gradient 2-70% B in 7 minutes

UV/Vis 214 nm

Results are summarized in Table 9. The concentration of CaCl₂ impacted results dramatically. An increase in digestion was observed using concentrations of CaCl₂ as high as 500 mM.

Table 9: Screening effect of buffer composition on digestion in solution

HEPES

758959 1758816 2816170 (50 m VI)

CaCl₂ Peptide Digested Undigested

0.05M 860714 2007752 2746822

0.1M 1074482 2377399 2139571

0.25M 1430859 3241428 2885626

0.5M 1474912 3340960 856015

1M 1344334 3203158 949033

Tris Peptide Digested Undigested

0.05M 860551 1995427 3125626

0.1M 1101647 2524942 2122289

0.25M 1564913 3462464 997441

0.5M 1297337 3218135 780101

1M 1431917 3458079 1156801

NaCl Peptide Digested Undigested

0.1M 648564 1527956 3295178

0.25M 484049 1178089 3432057

0.5M 350436 884946 4517343

1M 249834 670170 4981512

2M 111600 378641 4745627

_PH Peptide Digested Undigested

7 569651 1378134 4194144

7.5 558994 1300186 4135750

8 648093 1576109 3769910 8.5 1209006 2736695 1432732

9 1501708 3110740 733513

Gdn HC1⁴ Peptide Digested Undigested

0.5M 101735 216251 5487176

1M 94828 215746 5229914

2M 67546 184318 5455780

4M 11014 30954 6195166

6M 1647 5871 5301367

Urea Peptide Digested Undigested

0.5M 538728 1220686 3763696

1M 511139 1145096 2972094

2M 490181 1106227 3034093

4M 389932 860501 2877527

6M 301136 529552 3250299

8M 69027 156492 2197015

Glycerol Peptide Digested Undigested

5% 432166 1007768 3685707

10% 353626 833521 4404492

20% 275165 654553 5163829

OGS⁵ Peptide Digested Undigested

0.05% 581489 1424601 3798587

0.1% 675287 1616907 4128179

0.15% 636117 1538290 3751677 0.2% 619589 1574071 3732438

TWEEN Peptide Digested Undigested

0.01% 592916 1456333 4786849

0.05% 626837 1526614 5108006

0.1% 633934 1610365 5367425

Zwittergent Peptide Digested Undigested

0.1% 570687 1770158 3637403

0.2% 596902 1801486 5095067

0.5% 551913 1586693 5317700

1% 527021 1608067 6178885

SDC⁶ Peptide Digested Undigested

0.1% 5961 1284656 3123245

0.2% 480241 12332 29962

0.5% 361200 114517 499326

1% 239833 124131 1149062

DMSO⁷ Peptide Digested Undigested

5% 499051 1193067 3219771

10% 442885 1185116 3803031

15% 481657 1233021 4423224

20% 467559 1215388 4523186

ACN⁸ Peptide Digested Undigested

5% 625535 1409360 5143004 10% 692364 1676314 4742266

15% 779339 1925305 2715010

20% 1020020 2507679 3445401

MeOH⁹ Peptide Digested Undigested

5% 576911 1415794 5318134

10% 88569 216568 4800365

15% 666807 1700395 4218905

20% 756073 1986863 3394642

IPA¹⁰ Peptide Digested Undigested

5% 407876 1024085 3528233

10% 418703 1133300 3935383

15% 322103 906822 4509078

20% 347874 954036 4992140

Formamide Peptide Digested Undigested

5% 500704 1126069 3871868

10% 693291 1509456 4196754

15% 421130 973971 4281392

20% 194156 470938 4620417

TFE¹¹ Peptide Digested Undigested

5% 271046 710737 4417966

10% 201962 512715 4499811

15% 224520 571716 4785656

20% 253234 661078 4907160 1) ABC = ammonium bicarbonate 2) PBS = phosphate buffered saline 3) TBS = tris buffered saline 4) Gdn HC1 = guanidine hydrochloride 5) OGS = octylglucoside 6) SDC = sodium deoxycholate 7) DMSO = dimethylsulfoxide 8) ACN = acetonitrile 9) MeOH = methanol 10) IPA = isopropyl alcohol 11) TFE - tetrafluoroethylene

It is clear from these results that high concentrations of calcium chloride dramatically improve trypsin digestion using soluble trypsin. Also, glycerol in lower to moderate concentrations does not significantly, negatively impact digestion efficiency.

Example 10: Aggregation of various protein concentrations in optimized buffer at elevated temperatures in the presence of soluble trypsin

In order to determine the effect of the optimized buffer on aggregation various concentrations of bovine serum albumin were heated to 70°C in tris buffered saline, 500mM CaCl₂, 10% glycerol.. Trypsin was added in 1 :5 trypsin:protein ratio and the samples checked periodically for the formation of aggregates.

A summary of the results can be seen in Table 10 below.

Table 10: Summary of aggregation of various protein concentrations in optimized buffer at elevated temperatures in the presence of soluble trypsin

N= No aggregation

Y= Aggregation

S= Slight aggregation

60 N N N N

75 N N N N

90 N N N N

120 N N N N

180 N N N N

240 N N N N

The absence of aggregation in this example when using the optimized buffer, when compared to example 5, shows that the optimized buffer not only prevents aggregation when used in conjunction with a temperature stable immobilized enzyme, but also trypsin in solution. It was also surprising that the increase concentration of calcium did cause an increase in protein precipitation. Calcium is often removed from plasma by means of EDTA in order to prevent aggregation. In this case, when paired with glycerol, the presence of high concentrations of calcium chloride aids in the prevention of aggregation.

Example 11: Increased Calcium concentration has positive effect on protein digestion

Briefly, a temperature stable immobilized enzyme was prepared according to Example 1 and packed into a strip of 8 format according to Example 2. Samples were added directly to this slurry without pretreatment (neither reduction nor alkylation were employed). Samples were heated using a PCR thermo cycler. Following incubation the entire sample is removed using a multichannel pipette and transferred to a filter plate. A collection plate was placed below the filter plate and positive pressure was applied separating the immobilized enzyme from the digested sample.

Human, monoclonal IgGl was used in this experiment. The digestion efficiency was measured via measurement of peak area corresponding to the two peptides VVSVLTVLHQDWLNGK (SEQ ID NO 1) and TTPPVLDSDGSFFLYSK (SEQ ID NO 2). These two peptides are unique to human IgG. Table 11 shows the two different IgG peptides that are digested by trypsin and how they responded to various CaCl₂ concentrations in the reaction buffer. Table 11: Exemplary human IgG digestion efficiency in response to calcium concentration

75 1670 2097 2836 3869 3313 3195 2849 2192

90 1821 2083 2972 3844 N/A 3266 2901 2315

In this example we have shown that increasing the concentration of CaCl₂ beyond the established concentration range can have a dramatic impact on analyte response and enzyme activity. This is contrary to the established literature which says that when adding calcium chloride to tris buffer, "calcium chloride used in concentrations above ImM has shown no additional benefit in improving enzyme stability" (Sipos and Merkel. 1970. An Effect of Calcium Ions on the Activity, Heat Stability, and Structure of Trypsin. Biochemistry 9:2766- 2775), we see here that the increased concentration of calcium chloride above 1 mM has been shown to increase enzyme efficiency, measured by the two peak peptides unique to human IgG digestion by trypsin. In line with Sipo's findings many protocols recommend lmM-20mM CaCl₂ in tris buffer for trypsin digestion (Promega Technical Manual # 9PIV511, Depuydt et. al. 2013, Klee et. al. 2012, Liu et. al. 2014, Principe et. al. 2012, Yu et. al. 2011), but it is obvious such low concentrations of calcium are insufficient for enzyme digestions under these conditions.

Following Examples 12-14, further indicate that presence of glycerol or other sugar alcohol in the calcium containing reaction buffer may prevent protein aggregation in immobilized enzyme reactors.

Example 12: Quantitation of human IgG in monkey plasma

It was observed that when operated at high protein loads denaturation of proteins in the working solution could lead to aggregation. In these cases it was determined that the addition of glycerol to a 10% final concentration and use of 500 mM CaCl₂ prevented the formation of aggregates while enabling rapid digestion under denaturing conditions. The results below demonstrate quantitation of human IgG in 50 uL monkey plasma diluted in 150 uL of 50 mM TBS, 500 mM CaCl₂, 10% glycerol, digestion by temperature stable immobilized enzyme prepared according to Example 1 that was packed in a strip of 8 format prepared according to Example 2. Digestion occurred in 75 minutes using a ThermoMixer C operated at 70°C. Table 12: Sample preparation and LC/MS parameters for the digestion and analysis of human IgGi in 50uL monkey plasma

Reaction Conditions

Equipment Eppendorf ThermoMixer C

Sample Varying concentrations human IgG in monkey plasma

Digest Settings 70°C, 1600 RPM

Time 75 minutes

Digest Buffer 50mM TBS, 500mM CaCl₂, 10% glycerol

Diluent 50mM TBS, lOOmM CaCl₂

Digest to Diluent ratio 1 :99

Resin Amount 15uL

LCMS Conditions Used for Analysis

Injection Volume 5 uL of diluted sample Sample Hu IgGi

Reversed Phase A 2% ACN (aq) 0.1% Formic Acid

Reversed Phase B 90% ACN (aq) 0.1% Formic Acid

Reversed Phase Gradient 2-70%B in 5 minutes at 500uL/min

TTPPVLDSDGSFFLYSK (SEQ. ID. NO. 2) -

Peptide Sequence - MS1/MS2 937.74/836.43

Table 13: Calibration curves of human IgGl in monkey plasma

Table 14: Replicate analyses of human IgGl in monkey plasma at various concentrations

Example 13: Quantitation of human IgG in mouse plasma

The results below demonstrate quantitation of human IgG in 50uL mouse plasma diluted in 150 uL of 50 mM TBS, 500 mM CaCl₂, 10% glycerol, digestion by immobilized enzyme prepared according to Example 1 that was packed in a strip of 8 format prepared according to Example 2. Digestion occurred in 75 minutes using a ThermoMixer C operated at 70°C. Table 15: Sample preparation and LC/MS parameters for the digestion and analysis of human IgGi in 50uL monkey plasma

Table 16: Calibration curves of human IgGl in mouse plasma

Table 17: Replicate analyses of human IgGl in mouse plasma at various concentrations

Example 14: Quantitation of human IgG in beagle plasma

The results below demonstrate quantitation of human IgG in 50 uL beagle plasma diluted in 150 uL of 50 mM TBS, 500mM CaCl₂, 10% glycerol, digestion by immobilized enzyme prepared according to Example 1 that was packed in a strip of 8 format prepared according to Example 2. Digestion occurred in 75 minutes using a ThermoMixer C operated at 70°C.

Table 18: Sample preparation and LC/MS parameters for the digestion and analysis of human IgGi in 50uL beagle plasma

Reaction Conditions

Equipment Eppendorf ThermoMixer C

Sample Varying concentrations human IgG in beagle plasma

Digest Settings 70°C, 1600 RPM

Time 75 minutes

Digest Buffer 50mM TBS, 500mM CaCl₂, 10% glycerol

Diluent 50mM TBS, lOOmM CaCl₂

Digest to Diluent ratio 1 :99

Resin Amount 15uL

LCMS Conditions Used for Analysis

Injection Volume 5 uL of diluted sample Sample Hu IgGi

Reversed Phase A 2% ACN (aq) 0.1% Formic Acid

Reversed Phase B 90% ACN (aq) 0.1% Formic Acid

Reversed Phase Gradient 2-70%B in 5 minutes at 500uL/min

TTPPVLDSDGSFFLYSK (SEQ. ID. NO. 2) -

Peptide Sequence - MS1/MS2 937.74/836.43

Table 19: Calibration curves of human IgGl in beagle plasma

Table 20: Replicate analyses of human IgGl in beagle plasma at various concentrations

IgG in Beagle Plasma

Final IgG Concentration (ng/mL)

10 100 1000

Sample 1 108 917 9204

Sample 2 92 888 9565

Sample 3 99 938 9365

Sample 4 94 957 9788

Sample 5 73 950 8903

Sample 6 72 918 9751

Sample 7 914 9384

973 9746

Sample 8 90

Average 90 932 9463 StDev 13 28 311

CV(%) 14.7 3 3.3

Example 15: Comparing digestion buffers - solution digestion

Comparisons of digestion buffers used when performing traditional solution based digestion protocols were made using insulin as a model protein. Digestion was performed using a commercially available porcine trypsin. Briefly, a 5mg sample of USP standard human insulin was dissolved in 2% acetic acid, 100 mM glycine to a concentration of 10 mg/mL. This solution was then diluted 50 fold, to 200ug/mL, into digest buffer. The digest buffer varies according to the descriptions in Table 21. Starting with an 80ug/mL solution of trypsin in 20mM acetic acid, trypsin was added to achieve a 1 :25 enzyme: protein ratio. Each sample was digested for 18 hours at 37°C using a Veriti Thermo Cycler. Following digestion, lOOuL of sample was removed and acidified, quenching the reaction.

Table 21 summarizes conditions used for the analysis of insulin samples digested in various buffers.

Table 22 summarizes the effect of buffer composition on digestion in solution

Figure 3 provides a visual representation of the difference in peptide generation as a function of buffer composition when performing traditional solution based digestion protocols

Table 21: Conditions used for the analysis of insulin samples digested in various buffers

Table 22: Screening effect of buffer composition on digestion in solution

The increase in digestion products and decrease in undigested materials suggests that when performing traditional solution based digestion protocols enzyme activity associated with the use of ammonium bicarbonate is significantly higher than enzyme activity associated with the use of tris buffer containing calcium chloride.

Example 16: Comparing digestion buffers - digestion in an immobilized enzyme reactor

Comparisons of digestion buffers used when performing digestions in an immobilized enzyme reactor were made using insulin as a model protein. Briefly, digestion was performed using an immobilized enzyme prepared according to Example 1 packed in to a strip of 8 format prepared according to Example 2. A 5mg sample of USP standard human insulin was dissolved in 2% acetic acid, 100 mM glycine to a concentration of 10 mg/mL. This solution was then diluted 100 fold into the digest buffer of interest. The digest buffer varies according to the descriptions in Table 23. 200uL of this sample was added to each of 2 wells. Each sample was digested for 60 seconds at 70°C using a Veriti Thermo Cycler. The samples were then removed from the instrument and allowed to cool for 5 minutes. Once all of the samples had cooled the 2 wells of sample from each buffer condition were pooled, centrifuged and the digest materials decanted.

Table 23 summarizes conditions used for the analysis of insulin samples digested in various buffers in an immobilized enzyme reactor

Table 24 summarizes the effect of buffer composition on digestion in immobilized enzyme reactor Figure 4 provides a visual representation of the difference in peptide generation as a function of buffer composition when performing digestion in immobilized enzyme reactor

Table 23: Conditions used for the analysis of insulin samples digested in various buffers in an immobilized enzyme reactor

Table 24: Screening effect of buffer composition on digestion in immobilized enzyme reactor

The increase in digestion products and decrease in undigested materials suggests that when performing digestions in an immobilized enzyme reactor enzyme activity associated with the use of tris buffer containing calcium chloride is significantly higher than enzyme activity associated with the use of ammonium bicarbonate.

Example 17: Various concentrations of CaC12 in volatile buffers

As previously described, it was determined that the use of a tris buffer and high concentrations of CaC12 in combination with glycerol enables the digestion of 4x diluted plasma at elevated temperatures (PCT US62/025,783). In order to improve the mass spectrometric compatibility immobilized enzyme reactor working system, further experiments were performed using ammonium bicarbonate containing various concentrations of CaC12. However, these experiments resulted in the formation of a precipitate. As an alternative various concentrations of CaC12 were mixed with ammonium acetate. No precipitate was formed. Enzyme activity was determined using insulin as a model protein. Briefly, digestion was performed using an immobilized enzyme prepared according to Example 1 packed in to a strip of 8 format prepared according to Example 2. A 5mg sample of USP standard human insulin was dissolved in 2% acetic acid, 100 mM glycine to a concentration of 10 mg/mL. This solution was then diluted 100 fold into the digest buffer of interest. The digest buffer varies according to the descriptions in Tables 26 and 27. 200uL of this sample was added to each of 2 wells. Each sample was digested for 60 seconds at 70°C using a Veriti Thermo Cycler. The samples were then removed from the instrument and allowed to cool for 5 minutes. Once all of the samples had cooled the 2 wells of sample from each buffer condition were pooled, centrifuged and the digest materials decanted.

Table 25 summarizes conditions used for the analysis of insulin samples digested in various buffers, at various temperatures in an immobilized enzyme reactor

Table 26 summarizes peak areas associated with the effect of CaC12 concentration on digestion using ammonium acetate buffer in an immobilized enzyme reactor

Table 27 summarizes peak ratios associated with the effect of CaC12 concentration on digestion using ammonium acetate buffer in an immobilized enzyme reactor

Figure 5 provides a visual representation of precipitate formation associated with 50 mM ammonium bicarbonate, 1M CaC12 that has been heated to 70°C

Figure 6 provides a visual representation of precipitate formation associated with 50 mM ammonium bicarbonate, 5mM CaC12 that has been heated to 70°C

Figure 7 provides a visual representation of the effect of CaC12 concentration on digestion using ammonium acetate buffer in an immobilized enzyme reactor

Table 25: Conditions used for the analysis of insulin samples digested in 50mM ammonium acetate containing various concentrations of CaC12 Sample lOOug/mL digested Hu insulin

Injection Volume 25uL

Digest Conditions 70°C, 60 seconds

Mobile Phase A 2% ACN (aq) 0.1% Trifiuoroacetic Acid

Mobile Phase B 90% ACN (aq) 0.1% Trifiuoroacetic Acid

Reversed Phase Gradient 2-70% B in 7 minutes

UV/Vis 214 nm

Table 26: Screening effect of CaC12 concentration on digestion using ammonium acetate buffer in an immobilized enzyme reactor (peak areas)

1) AA = ammonium acetate, 2) CaC12 = Calcium Chloride

In order to normalize for variations in total protein recovery, ratios of the digestion product were also calculated by taking the area of a given peak and dividing this peak are by the sum of all of the peaks.

Table 27: Screening effect of CaC12 concentration on digestion using ammonium acetate buffer in an immobilized enzyme reactor (peak ratios)

50mM ABC, 50mM CaC12 Not Applicable , Precipitate Formed

50mM ABC, lOOmM CaC12 Not Applicable , Precipitate Formed

50mM ABC, 250mM CaC12 Not Applicable , Precipitate Formed

50mM ABC, 500mM CaC12 Not Applicable , Precipitate Formed

50mM ABC, lOOOmM CaC12 Not Applicable , Precipitate Formed

50mM AA, OM CaC12 361466 808724 4051529 5221719

50mM AA, 5mM CaC12 770962 1813176 2204667 4788805

50mM AA, 50mM CaC12 652457 1477943 2589951 4720351

50mM AA, lOOmM CaC12 689448 1576369 3111616 5377433

50mM AA, 250mM CaC12 590992 1355891 2903737 4850620

50mM AA, 500mM CaC12 545619 1273996 3334547 5154162

50mM AA, lOOOmM CaC12 413165 917615 2913292 4244072

Example 18: Comparing digestion buffers continued - digestion in an immobilized enzyme reactor

Comparisons of digestion buffers used when performing digestions in an immobilized enzyme reactor were made using insulin as a model protein. Briefly, digestion was performed using an immobilized enzyme prepared according to Example 1 packed in to a strip of 8 format prepared according to Example 2. A 5mg sample of USP standard human insulin was dissolved in 2% acetic acid, 100 mM glycine to a concentration of 10 mg/mL. This solution was then diluted 100 fold into the digest buffer of interest. The digest buffer varies according to the descriptions in Table 29. 200uL of this sample was added to each of 2 wells. Each sample was digested for 60 seconds at 70°C using a Veriti Thermo Cycler. The samples were then removed from the instrument and allowed to cool for 5 minutes. Once all of the samples had cooled the 2 wells of sample from each buffer condition were pooled, centrifuged and the digest materials decanted.

Table 28 summarizes conditions used for the analysis of insulin samples digested in various buffers in an immobilized enzyme reactor Table 29 summarizes the effect of buffer composition on digestion in immobilized enzyme reactor

Figure 8 provides a visual representation of the difference in peptide generation as a function of buffer composition when performing digestion in immobilized enzyme reactor

Table 28: Conditions used for the analysis of insulin samples digested in various buffers in an immobilized enzyme reactor

Table 29: Screening effect of buffer composition on digestion in immobilized enzyme reactor

The increase in digestion products and decrease in undigest materials suggests that when performing digestions in an immobilized enzyme reactor enzyme activity associated with the use of 50 mM ammonium acetate containing calcium chloride is significantly higher than enzyme activity associated with the use of ammonium bicarbonate or the use of calcium chloride alone. However, the use of immobilized enzyme reactor enzyme activity associated with the use of 50 mM sodium acetate containing calcium chloride was comparable to that of 50 mM ammonium acetate containing calcium chloride.

As Table 29 indicates, digestion efficiency increases dramatically in the presence of acetate and calcium chloride. These increases were not observed in solutions containing only calcium chloride or in solutions containing ammonium ions. Although it is not clear what makes the combination of acetate ions and calcium chloride associate with higher trypsin digestion efficiency, it is clear that this combination has a synergistic effect on trypsin digestion.

It is contemplated that other acetate solutions may have a similar effect on enzyme at elevated temperature. For example aluminum acetate, ammonium acetate, potassium acetate, propyl acetate, phenyl acetate, octyl acetate, dodecyl acetate, geranyl acetate, glycerin acetate, amyl acetate, vinyl acetate, methyl acetate, ethyl acetate, isopropyl acetate, ethylhexyl acetate, butyl acetate, sodium acetate, copper acetate, calcium magnesium acetate, cesium acetate, barium acetate, beryllium acetate, cadmium acetate, magnesium acetate, chromium acetate, iron acetate, lead acetate, manganese acetate, sodium diacetate, lithium acetate, pyridine acetate, mercury acetate, molybdenum acetate, nickel acetate, palladium acetate, platinum acetate, rhodium acetate, silver acetate, triethylammonium acetate, trimethylammonium acetate and zinc acetate solutions are expected to show temperature dependent increases in enzyme reaction. It is further contemplated that other volatile salts may have a similar effect on enzyme at elevated temperature. For example ammonium acetate, ammonium formate, pyridine acetate, N- ethylmorpholine acetate, triethylammonium acetate and trimethylammonium acetate solutions are expected to show temperature dependent increases in enzyme reaction. It is further contemplated that other metal ions may have a similar effect on enzyme at elevated temperature. For example, Na⁺, Mg²⁺, Ca²⁺, Sr²⁺, Mn²⁺, Co²⁺, Ni²⁺, Zn²⁺, Cu²⁺, Fe²⁺ , Fe³⁺ in various acetate solutions are expected to show temperature dependent increases in enzyme reaction. One non- limiting explanation is that the combined presence of both acetate and metal ions in the reaction buffer, especially in the IMER format enzyme reactions, dramatically increases the chances of enzyme to contact substrate by unknown means. Another non-limiting explanation of the combined acetate metal effect in the reaction buffer is that the enzyme may be transformed by the presence of these ions to become a more active format, such as undergoing conformational change to have altered Kcat/Km ratio that favors higher reaction efficiency. Regardless, it is a surprising discovery from this disclosure that the combination of acetate and metal ions can be revised further from the currently available art and protocols to improve the immobilized enzyme reaction efficiency at elevated temperatures.

Example 19: Buffer effects of protein digestion in immobilized enzyme reactor at various temperatures

Briefly, digestion was performed using an immobilized enzyme prepared according to Example 1 packed in to a strip of 8 format prepared according to Example 2. A 5mg sample of USP standard human insulin was dissolved in 2% acetic acid, 100 mM glycine to a concentration of 10 mg/mL. This solution was then diluted 100 fold into the digest buffer of interest. The digest buffer varies according to the descriptions in Table 31. 200uL of this sample was added to each of 2 wells. Each sample was digested for 120 seconds at various temperatures using a Veriti Thermo Cycler. The sample was then removed from the instrument and allowed to cool for 5 minutes. Once all of the samples had cooled the 2 wells of sample from each buffer condition were pooled, centrifuged and the digest materials decanted.

Table 30 summarizes conditions used for the analysis of insulin samples digested in various buffers, at various temperatures in an immobilized enzyme reactor

Table 31 summarizes the effects of buffer composition on digestion in an immobilized enzyme reactor at various temperatures

Figure 9 provides a visual representation of the effect of buffer composition on digestion in an immobilized enzyme reactor at various temperatures

Table 30: Conditions used for the analysis of insulin samples digested in various buffers, at various temperatures in an immobilized enzyme reactor

Mobile Phase B 90% ACN (aq) 0.1% Trifiuoroacetic Acid

Reversed Phase Gradient 2-70% B in 7 minutes

UV/Vis 214 nm

Table 31: Screening effect of buffer composition on digestion in an IMER at various temperatures

1) ABC = ammonium bicarbonate 2) AA = ammonium acetate 3) calcium chloride

Example 20: Protein aggregation at various concentrations associated with the use of ammonium acetate buffer containing SmM CaC12

In order to determine the effect of the optimized buffer on aggregation, various concentrations of bovine serum albumin were heated to 70°C in 50mM ammonium acetate, 5mM CaCl. A temperature stable immobilized enzyme was prepared according to Example 1 and packed into a strip of 8 format according to Example 2. Samples were added directly to this slurry without pretreatment (neither reduction nor alkylation were employed). Samples were heated using a ThermoMixer C operated at 70°C and 1300rpm and the samples were checked periodically for the formation of aggregates.

A summary of the results can be seen in Table 32 below.

Table 32 Aggregation of various concentrations of BSA in an IMER utilizing ammonium acetate buffer containing 5mM CaC12

N= No aggregation

Y= Aggregation

S= Slight aggregation

Example 21: Protein aggregation at various concentrations associated with the use of ammonium acetate buffer containing 5mM CaC12, 0.1% octylglucoside

In order to determine the effect of the optimized buffer on aggregation various concentrations of bovine serum albumin were heated to 70°C in 50mM ammonium acetate, 5mM CaCl, 0.1% octylglucoside. A temperature stable immobilized enzyme was prepared according to Example 1 and packed into a strip of 8 format according to Example 2. Samples were added directly to this slurry without pretreatment (neither reduction nor alkylation were employed). Samples were heated using a ThermoMixer C operated at 70°C and 1300rpm and the samples were checked periodically for the formation of aggregates.

A summary of the results can be seen in Table 33 below.

Table 33 Aggregation of various concentrations of BSA in an IMER utilizing ammonium acetate buffer containing 5mM CaC12 and 0.1% octylglucoside

N= No aggregation

Y= Aggregation

S= Slight aggregation

90 N N N N N N Y

120 N N N N N S Y

180 N N N N N S Y

240 N N N N N Y Y

As Table 32 indicates, the presence of octylglucoside in the optimized buffer enhances sample solubility and prevents aggregation. It is contemplated that other surfactants or detergents may have a similar effect on aggregation at elevated temperature. For example octylglucoside, sodium dodecyl sulfate, sodium laureth sulfate, ammonium lauryl sulfate, sodium pareth sulfate, Big CHAP deoxy, ASB, sodium deoxycholate, or acid-cleavable detergents are expected to prevent protein aggregation during digestion at elevated temperatures.

Example 22: Protein aggregation at various concentrations associated with the use of ammonium acetate buffer containing 5mM CaC12, 5% glycerol

In order to determine the effect of the optimized buffer on aggregation various concentrations of bovine serum albumin were heated to 70°C in 50mM ammonium acetate, 5mM CaCl, 5% glycerol. A temperature stable immobilized enzyme was prepared according to Example 1 and packed into a strip of 8 format according to Example 2. Samples were added directly to this slurry without pretreatment (neither reduction nor alkylation were employed). Samples were heated using a ThermoMixer C operated at 70°C and 1300rpm and the samples were checked periodically for the formation of aggregates.

A summary of the results can be seen in Table 34 below.

Table 34 Aggregation of various concentrations of BSA in an IMER utilizing ammonium acetate buffer containing 5mM CaC12 and 5% glycerol

N= No aggregation

Y= Aggregation

S= Slight aggregation

90 N N N N N N s

120 N N N N N N Y

180 N N N N N N Y

240 N N N N N Y Y

Example 23: Protein aggregation at various concentrations associated with the use of ammonium acetate buffer containing 5mM CaC12, 10% glycerol

In order to determine the effect of the optimized buffer on aggregation various concentrations of bovine serum albumin were heated to 70°C in 50mM ammonium acetate, 5mM CaCl, 10% glycerol. A temperature stable immobilized enzyme was prepared according to Example 1 and packed into a strip of 8 format according to Example 2. Samples were added directly to this slurry without pretreatment (neither reduction nor alkylation were employed). Samples were heated using a ThermoMixer C operated at 70°C and 1300rpm and the samples were checked periodically for the formation of aggregates.

A summary of the results can be seen in Table 35 below.

Table 35 Aggregation of various concentrations of BSA in an IMER utilizing ammonium acetate buffer containing 5mM CaC12 and 10% glycerol

N= No aggregation

Y= Aggregation

S= Slight aggregation

50 m VI Am. Ac.

w/ m VI CaC12 Concentration of BSA (mg/mL)

and 10% glycerol @ 70C

1.25 2.5 3.75 6.25 12.5 25 37.5

5 N N N N N N N

10 N N N N N N N

15 N N N N N N N

30 N N N N N N Y

45 N N N N N N Y

Incubation Time

60 N N N N N N Y

75 N N N N N N Y

90 N N N N N N Y

120 N N N N N N Y

180 N N N N N N Y

240 N N N N N N Y

As Tables 34 and 35 indicate, the presence of glycerol in the optimized buffer enhances sample solubility and prevents aggregation. It is contemplated that other sugar alcohols or sugars may have a similar effect on aggregation at elevated temperature. For example glycol, trehalose, glucose, fructose, sucrose, maltose, lactose, galactose, allose, altrose, mannose, gulose, idose, talose, sorbitol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol or inositol are expected to prevent protein aggregation during digestion at elevated temperatures.

It should be understood that the foregoing relates to exemplary embodiments of the disclosure and that modifications may be made without departing from the spirit and scope of the invention as set forth in the following claims.

Claims

CLAIMS:

1. An improved enzyme reactor system that prevents aggregation at an elevated temperature, comprising essentially the following components:

a. an enzyme reactor that provides rapid hydrolysis to at least one substrate, wherein said hydrolysis utilizes an enzyme selected from the group consisting of trypsin, chymotrypsin, Lys-C, Glu-C, Arg-C, Asp N, papain, pepsin, elastase, IdeS, pronase and PNGase F and a combination thereof; and

b. a reaction buffer comprising sugar alcohols or sugars selected from the group consisting of glycerol, glycol, trehalose, glucose, fructose, sucrose, maltose, lactose, galactose, allose, altrose, mannose, gulose, idose, talose, sorbitol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol and inositol.

2. The improved enzyme reactor in claim 1, further comprising metal cation concentration greater than 1 nanomolar in said reaction buffer, wherein said metal cation in the reaction buffer is selected from the group consisting of aluminum, calcium, copper, iron, magnesium, manganese, mercury, sodium and silver.

3. An improved enzyme reactor system comprising an immobilized enzyme and an enzyme reaction buffer that is compatible with mass spectrometric analyses, said system enables enhanced enzymatic digestion and prevents protein aggregation at elevated temperatures , wherein said reaction buffer comprises an acetate salt in combination with metal concentration greater than 1 nanomolar.

4. An improved enzyme reactor system comprising an immobilized enzyme and an enzyme reaction buffer that is compatible with mass spectrometric analyses, said system enables enhanced enzymatic digestion and prevents protein aggregation at elevated temperatures at elevated temperatures, wherein said reaction buffer comprises a volatile salt in combination with metal concentration greater than 1 nanomolar.

5. The improved enzyme reactor system in claim 3,

wherein the enzyme is selected from the group consisting of trypsin, chymotrypsin, Lys-C, Glu-C, Arg-C, Asp N, papain, pepsin, elastase, IdeS, pronase and PNGase F or a combination thereof; wherein the reaction buffer comprising acetate salts selected from the group consisting of aluminum acetate, ammonium acetate, potassium acetate, propyl acetate, phenyl acetate, octyl acetate, dodecyl acetate, geranyl acetate, glycerin acetate, amyl acetate, vinyl acetate, methyl acetate, ethyl acetate, isopropyl acetate, ethylhexyl acetate, butyl acetate, sodium acetate, copper acetate, calcium magnesium acetate, cesium acetate, barium acetate, beryllium acetate, cadmium acetate, magnesium acetate, chromium acetate, iron acetate, lead acetate, manganese acetate, sodium diacetate, lithium acetate, pyridine acetate, mercury acetate, molybdenum acetate, nickel acetate, palladium acetate, platinum acetate, rhodium acetate, silver acetate, triethylammonium acetate, trimethylammonium acetate, zinc acetate; wherein the metal cation in the reaction buffer is selected from the group consisting of aluminum, calcium, copper, iron, magnesium, manganese, mercury, sodium and silver.

6. The improved enzyme reactor system in claim 4, wherein said enzyme is selected from the group consisting of trypsin, chymotrypsin, Lys-C, Glu-C, Arg-C, Asp N, papain, pepsin, elastase, IdeS, pronase and PNGase F or a combination thereof; wherein the reaction buffer comprising volatile salts selected from the group consisting of ammonium acetate, ammonium formate, pyridine acetate, N-ethylmorpholine acetate, trimethylammonium acetate, triethylammonium acetate; and wherein the metal cation in the reaction buffer is selected from the group consisting of aluminum, calcium, copper, iron, magnesium, manganese, mercury, sodium and silver.

7. The reaction buffer in any of claims 1, 3, or 4, wherein said reaction buffer is further comprised of either a surfactant, or detergent selected from the group consisting of octylglucoside, sodium dodecyl sulfate, sodium laureth sulfate, ammonium lauryl sulfate, sodium pareth sulfate, Big CHAP deoxy, ASB, sodium deoxycholate, an acid-cleavable detergent or a combination thereof.

8. The reaction buffer in any of claims 3 or 4, wherein said reaction buffer is further comprised of either a sugar alcohol or sugar selected from the group consisting of glycerol, glycol, trehalose, glucose, fructose, sucrose, maltose, lactose, galactose, allose, altrose, mannose, gulose, idose, talose, sorbitol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol and inositol or a combination thereof.

9. The improved enzyme reactor system in any of claims 1, 3 or 4 wherein operation is performed at elevated temperature of 37°C or above.

10. The improved enzyme reactor system in any of claims 1, 3 or 4 wherein said enzyme is a modified enzyme selected from the group consisting of modified trypsin, chymotrypsin, Lys- C, Glu-C, Arg-C, Asp N, papain, pepsin, elastase, IdeS, pronase and PNGase F or a combination thereof.

11. The improved enzyme reactor system in claim 1 , wherein said hydrolysis is conducted by an enzyme in solution, an immobilized enzyme reactor, microwave reactor, ultrasound, infrared, filter aided sample preparation, solvent aided trypsin digestion, a pressure reactor or any combination thereof.

12. The improved enzyme reactor system in any of claims 3 or 4, wherein said hydrolysis is conducted in a microwave reactor, ultrasound, infrared, filter aided sample preparation, solvent aided trypsin digestion, a pressure reactor or any combination thereof.

13. The improved enzyme reactor system in any of claims 1, 3 or 4 wherein said reaction buffer formulations further comprise co-solvents including but not limited to organic solvents, chaotropes, surfactants, detergents, salts, sugars, sugar alcohols, and any combination thereof.

14. The improved enzyme reactor system in any of claims 1, 3 or 4, wherein the enzyme's substrate is not denatured, reduced, alkylated either before or after hydrolysis.

15. The improved enzyme reactor system in any of claims 1, 3 or 4, wherein the enzyme's substrate is denatured, reduced, alkylated either before or after hydrolysis.

16. The improved enzyme reactor system in any of claims 1, 3 or 4, wherein said reactor format is a combination heater/shaker instrument, a heating block on a shaker, shaking in a convection oven, shaking in a water bath, shaking in an incubator, shaking in a microwave oven, a heater, a heating block, a convection oven, a water bath, an incubator, a microwave oven, or any combination thereof.

17. The improved enzyme reactor system in any of claims 1, 3 or 4, wherein said reaction is carried out a thin walled PCR tube, any thin walled sample tube, or multi-well plate.

18. The improved enzyme reactor system in any of claims 1, 3 or 4, wherein said reactor is in the form of a column, eppendorf tube, pipette tip, multi well plate, or magnetic bead.

19. The improved enzyme reactor system in any of claims 3 or 4, wherein the immobilized enzyme having a supporting material selected from the group consisting of polystyrene, polystyrene/divinylbenzene, silica, controlled porosity glass, dextrans, agarose, acrylates, magnetic support materials and nitrocellulose.

20. The improved enzyme reactor system in claim 19, wherein said immobilized enzyme supporting material is in a form of a particle, magnetic particle, monolithic, membrane, planar or micro fluidic channel.