GENERAL

The methodological approaches used for the microextraction and purification of amyloid proteins vary in different laboratories, and sometimes it might be unclear (especially for the researcher less experienced in this specific field) how to select the appropriate methodology for the case under study. This is in contrast to classic large scale techniques, where there are clear cut guidelines for amyloid protein purification. Therefore, to overcome this problem, we have classified the existing microtechniques—first, according to the extraction solvent used, and second, according to the purification technique applied. Furthermore, the advantages and disadvantages of these new microtechniques have been outlined by taking into consideration the tissue type (formalin fixed or unfixed) and size, amyloid type, and amyloid content in the sample.

MICROEXTRACTION

Fresh tissue

Several microextraction procedures have been developed and applied to recover amyloid proteins from milligram to gram quantities of fresh frozen tissue. Tissues were usually homogenised and washed with buffered saline before the extraction procedure; in some cases, tissue homogenates were digested with collagenase and DNase^11,12 or defatted.^13–16 A rapid water extraction procedure was described and used to obtain amyloid fibrils in their native state.¹⁷ In contrast, other micromethods have been developed that use denaturing conditions. Because most amyloid proteins are soluble in 6M guanidine hydrochloride solution, these proteins were frequently extracted using this denaturant.^{13–16,18,19} In several reports, formic acid was used to recover Aβ, AL, and ATTR from small fresh tissue specimens.^11,12,20 It was found that AA, AL, ATTR, and some more soluble Aβ species could also be extracted with a milder solvent, namely: 20% acetonitrile/0.1% trifluoroacetic acid (TFA).^1,12,21–34

Formalin fixed tissue

Formalin, like other aldehyde based fixatives, forms intermolecular and intramolecular crosslinks, which might complicate the subsequent extraction of proteins from fixed tissues. However, several extraction procedures have been developed and found to be helpful in overcoming the difficulties related to the high insolubility of proteins present in formalin fixed paraffin wax embedded tissues. Incubation of the dewaxed amyloid laden fixed tissues in a standard 3% sodium dodecyl sulfate (SDS) electrophoresis buffer at 90°C for five minutes led to the solubilisation of AA, but not other amyloid proteins, (such as AL, ATTR, and Aβ₂ microglobulin).^35,36 Extraction with 2.5% SDS and 8M urea containing electrophoresis sample buffer at 90°C for 15 minutes enabled the recovery of AL, Aβ, and ATTR proteins.^37,38 Because no proteins were detected when the same extraction technique was applied to amyloid free formalin fixed tissues, it was proposed that amyloids, in contrast to other tissue proteins, “survive” formalin fixation. The resistance of amyloids to crosslinking could be related to the deposition of amyloids in densely packed aggregates with a β pleated sheet conformation, which could prevent the infiltration of formalin and crosslinking.^37,38 However, other studies found that incubation of formalin fixed samples with 2% SDS containing buffers at high temperatures for a longer period of time resulted in the extraction of “non-amyloid” proteins, such as E-cadherin, β-catenin, and cyclin D1.³⁹ In fact, the recovery of these proteins increased significantly as the temperature increased: the highest recoveries were obtained by incubating the samples at 100°C for 20 minutes, followed by incubation at 60°C for two hours. It was suggested that the prolonged exposure of a fixed sample to high temperature might lead to the dissociation of crosslinking.³⁹ Thus, it appears that although the β pleated sheet configuration could be an important structural factor preserving the chemical integrity of amyloids, the experimental extraction conditions are crucial for the disruption of the existing crosslinks.

“The resistance of amyloids to crosslinking could be related to the deposition of amyloids in densely packed aggregates with a β pleated sheet conformation, which could prevent the infiltration of formalin and crosslinking”

In some other studies, formalin fixed tissues were extracted using either formic acid or guanidine salts. Incubation of the dewaxed fixed tissues with formic acid overnight at room temperature allowed the solubilisation of the Aβ⁴⁰ and AL^11,41 proteins. In a recent study performed with a large number of fixed specimens (n = 49), different types of amyloid proteins were extracted by incubation in 6M guanidine solutions over 24 to 72 hours at 37°C.⁴²

ELECTROPHORETIC SEPARATION AND ANALYSIS

SDS polyacrylamide gel electrophoresis (SDS-PAGE) based western blotting and microsequencing is a simple and very frequently used approach to separate and analyse the microextracted amyloid proteins. The amyloid material extracted either by a water extraction procedure or by SDS containing buffers can be directly applied to SDS gels; formic acid and acetonitrile/TFA tissue extracts have to be dried, redissolved in sample buffer, and then run electrophoretically. Guanidine extracted material must be extensively dialysed before SDS-PAGE (because SDS precipitates in the presence of guanidine salts). In fact, the purification of guanidine extracted amyloid proteins is performed mainly by chromatographic means, as described in the following section. Our experience working with fresh tissues showed that for direct electrophoretic examination of crude tissue extracts, volatile extraction solvents are preferable, especially when manipulating extremely small amounts of available tissue. Such solvents can be removed by lyophilisation or by Speed-Vac drying without the need for dialysis, and the proteins could be easily re-extracted, thus increasing the recovery of amyloid.

Fresh tissue

In some reports, western blotting and microsequencing techniques were effectively used to characterise amyloid recovered from tissues by formic acid extraction. ALλ-II deposits were identified in the ureteral tissue of a patient with localised ureteral amyloidosis.¹¹ TTR deposits of human origin were revealed in kidneys of mice transgenic for wild-type human transthyretin.¹²

Acetonitrile/TFA extraction followed by western blotting was used to determine the type of amyloid proteins present in very small amounts of diagnostic biopsy samples, including those of the spleen, liver, heart, thyroid, and skin²⁵ and in fine needle fat aspirates.³¹ Such methods might contribute to the precise diagnosis of disease, and are particularly useful in cases where the data obtained by common immunohistochemical techniques are unexpected or questioned. Western blot analysis of acetonitrile/TFA extracts obtained from milligram amounts of AD brain tissue was used for Aβ detection²⁶; this assay was used to distinguish between AD and other dementias.³⁰ The same microextraction technique was used in the examination of skin biopsy specimens taken from patients with localised skin amyloidosis,²⁸ a disorder where the chemical nature of the amyloid fibril protein has not yet been identified. The deposition of full length apolipoprotein E (apoE) and its fragment was revealed, indicating the importance of these minor components in amyloid formation. Western blot analysis of amyloids microextracted with acetonitrile/TFA was used to study biopsy specimens taken from patients with cardiac and larynx amyloidosis.³² This study revealed a rare case showing colocalisation of two different amyloid types—ATTR and a new variant of apoA-I. In studies where the acetonitrile/TFA extracts contained sufficient amounts of amyloid, the electrophoresed proteins were successfully sequenced after they were transferred to polyvinyl difluoride (PVDF) membranes.^12,23,27,29 Such a technique has been applied to study the fibrillar (amyloid) and non-fibrillar (non-amyloid) deposits coexisting in the same patient with B cell proliferative disease.²⁷ It was shown that both types of deposits had very similar molecular masses and identical N-terminal sequences belonging to the immunoglobulin light chain variable region of the κ-IV subgroup. The acetonitrile/TFA microextraction technique followed by western blotting,^12,23 amino acid sequencing,^12,23 and mass spectral analysis¹² has also been used to study small animal models of experimentally induced AA amyloidosis,²³ senile systemic amyloidosis,¹² and Down’s syndrome neuropathology (C Harris-Cerruti et al. Deleterious association of elevated CuZn superoxide dismutase activity and amyloid precursor protein in a transgenic mouse model of Down syndrome neuropathology. IXth International Symposium on Amyloidosis, Budapest, 2001, abstract 9.1.10). The data obtained in such studies led to several assumptions on the mechanisms involved in the formation of amyloid fibrils. Thus, in casein induced murine amyloidosis,²³ tissue deposition of both AA and its precursor the SAA2 isomer was revealed: SAA2 was detected only in the early stages of the amyloidogenic process, whereas substantial amounts of AA appeared later. It was assumed that in murine amyloidosis, the deposition of SAA2 proteins is an initial step in fibril formation, which is afterwards followed by SAA processing to AA. Similar to the case of combined light chain deposition disease and AL amyloidosis,²⁷ the presence of both fibrillar and non-fibrillar human TTR was revealed in mice transgenic for wild-type human TTR.¹² Both types of TTR deposits contained intact human TTR monomers with no evidence of proteolysis or co-deposition of murine TTR. It was found that the non-fibrillar proteins were deposited before the appearance of fibrils, thus suggesting that the non-amyloid deposits represent a precursor of the fibril.

Fixed tissue

Western blotting and microsequencing techniques were used to study amyloid proteins microextracted from fixed tissues with SDS containing buffers^36–38 and formic acid.^12,40,41 The analysis of formic acid extracted material allowed the identification of Aβ proteins in the brain of a patient with AD.⁴⁰ Formic acid extraction was also useful in identifying amyloid deposits consisting of λ-III immunoglobulin light chain fragments in ureteral tissue¹² and immunoglobulin light chain λ type deposits in a case of amyloidosis of the urinary bladder.⁴¹ In other studies, where fixed tissues were extracted with 2% SDS/8M urea containing buffers, amino acid sequence analysis revealed ALλ deposits in the leptomeningeal blood vessels of a patient with cerebral amyloidosis,³⁷ ATTR in the heart of a patient with systemic senile amyloidosis,³⁸ and AL-κ in the spleen of a patient with systemic amyloidosis.³⁸

CHROMATOGRAPHIC PURIFICATION AND ANALYSIS

Fresh tissue

Gel filtration has been used to purify amyloid proteins extracted with 6M guanidine from relatively larger biopsy specimens. Amyloid extracts obtained from subcutaneous biopsy specimens (about 1 cm³)^13,16 and from intracerebral “amyloidoma” (2 g)¹⁵ were separated on a Sephacryl S-200 ¹³ or a Sepharose 6B CL column.^15,16 Proteins were eluted with guanidine containing buffer, dialysed, lyophilised, and subjected to amino acid sequence analysis. These studies allowed the identification of the Alκ-I,¹³ ALλ (subgroup III or IV),¹⁴ and Alκ-IV¹⁶ proteins.

“Reversed phase high performance liquid chromatography has not gained wide application in the analysis of AA, AL, ATTR, and other proteins present in the tissues of patients with systemic amyloidosis”

Although reversed phase high performance liquid chromatography (HPLC) is efficiently used for the micropurification of proteins in different fields of biology and medicine, this technique has not gained wide application in the analysis of AA, AL, ATTR, and other proteins present in the tissues of patients with systemic amyloidosis. In fact, reversed phase HPLC on Lichrosorb RP-18 (10 μm particle size, 250 × 4.6 mm internal diameter; Knauer, Berlin, Germany) and on Vydac 218 TP (5 mm particle size, 300 Å pore diameter, 250 × 4.6 mm internal diameter; Alltech, Deerfield, Illinois, USA) columns was effective for the purification of AA^43,44 but not for AL or ATTR.^43,45 Reversed phase HPLC was used to purify Aβ proteins microextracted with formic acid from AD brain tissue.²⁰ In this study, tissue homogenates were washed with SDS containing buffers before Aβ extraction, so that the HPLC purified Aβ material represented the SDS insoluble Aβ fraction. In our study, which aimed to recover the total amount of Aβ deposits, the AD tissue homogenates were washed in the absence of this detergent and extracted using acetonitrile/TFA and formic acid sequentially.³⁴ Although the extracted material contained large amounts of Aβ proteins, the application of reversed phase HPLC alone was insufficient for the purification of these proteins (B Kaplan, 1999, unpublished data).

Size exclusion HPLC in SDS containing buffer (Bio-Sil TSK-125, 5 μm; Bio-Rad, Richmond, California, USA) was applied to study AA, AL, and ATTR proteins microextracted with acetonitrile/TFA.²¹ However, this technique resulted only in partial purification of amyloid proteins, as assessed from the electrophoretic examination of the HPLC fractions obtained. Efficient purification of low molecular mass amyloid (< 12 kDa), such as AA and small AL proteins, was achieved on a size exclusion HPLC column in aqueous organic solvent.⁴⁶ Using this technique, amyloid protens were microextracted with 20% acetonitrile/0.1% TFA from fresh frozen samples and further purified on a Bio-Sil TSK column equilibrated with the same solvent.²¹ Unfortunately, this technique was not useful for studying the higher molecular mass amyloid proteins (> 12 kDa). It should be noted that elution with acetonitrile/TFA resulted in an aberrant size exclusion behaviour on the Bio-Sil TSK column, presumably because of the interaction of the protein with the residual silanols on the silica based HPLC columns. This might explain why the larger amyloid proteins were poorly resolved by this technique.^21,46

Fixed tissue

In most studies, reversed phase HPLC was used for the purification of fixed amyloid proteins recovered from tissues by the guanidine extraction technique.^1,42 Briefly, amyloid proteins extracted with 6M guanidine hydrochloride containing buffer were reduced and alkylated, and then run on an Aquapore 300 C₈ (30 × 4.0 mm) column (Brownlee Column; Perkin Elmer, Norwalk, Connecticut, USA). The HPLC purified material was sequenced, thus allowing identification of nine types of amyloid proteins (including AA, AL, ATTR, Alys, and ApoA-I) found in different biopsy samples (for example, spleen, kidney, liver, heart, abdominal fat, and colon). Recently, this technique was used to study formalin fixed heart tissue samples from a patient with senile systemic amyloidosis, where ATTR was found to be co-deposited with a minor amyloid constituent, apoA-IV.⁴⁷ In this study, the ability of apoA-IV to enhance amyloid formation was demonstrated. Thus, guanidine extraction from formalin fixed tissues, followed by separation on reversed phase HPLC columns and chemical analysis of purified amyloid proteins, is a valuable technique for the precise diagnosis of amyloid associated disorders and could aid in the examination of the mechanisms of amyloid formation in these diseases.

COMBINED PURIFICATION TECHNIQUES

Although the number of separation steps should be as small as possible in small scale purification design, it is not always possible to purify microextracted amyloid proteins in a single separation step. We faced such a situation in the analysis of fresh frozen (non-fixed) tissue extracts where the content of amyloid proteins was small and the contamination by other co-extracted tissue proteins was great.⁴⁵ In fact, micropreparative slab gel SDS electrophoresis allows the efficient separation of proteins on the basis of their differences in molecular mass, but in using this method separated amyloid proteins might still be contaminated by other proteins of the same electrophoretic mobility. In cases where the amount of the available biopsy material was sufficient (such as subcutaneous fat biopsies^13,14), purification was achieved by combining gel filtration on a 800 × 9 mm internal diameter Sephacryl S-2000 HR (Pharmacia, Uppsala, Sweden) column with reversed phase HPLC on a 250 × 4.6 mm VYDAC 214 TPS (Hesperia, California, USA) column. However, such relatively large gel filtration columns are less practical when analysing the extracts obtained from smaller tissue samples. Although the use of size exclusion HPLC columns could be more appropriate for small samples, our experience showed that in many instances the resolution of amyloid proteins obtained by size exclusion HPLC was less efficient than when micropreparative slab gel SDS electrophoresis was used.⁴⁵

We developed a technique that combines slab gel SDS electrophoresis (SDS-PAGE) with subsequent reversed phase HPLC,^44,48,49 and recently adapted it for the purification of AA, AL, ATTR, and Aβ proteins microextracted from non-fixed tissues.^33,34,50 Using this technique, the electrophoresed proteins were transferred to PVDF membranes, then eluted from the membranes, and finally applied to reversed phase HPLC. We found that the elution from PVDF with TFA/acetonitrile/water (3/4/3, vol/vol) solvent allowed efficient recovery of the AA, AL, ATTR, and Aβ proteins from the PVDF membranes, and was compatible with the subsequent reversed phase HPLC separation. A consecutive SDS-PAGE and reversed phase HPLC technique was used for the purification of Aβ from crude AD brain tissue extracts.^34,50 It allowed the amyloid proteins to be separated electrophoretically from higher and lower molecular mass tissue components, and then purified by reversed phase HPLC from the contaminants that had similar molecular masses but different retention times on the column.

CONCLUDING REMARKS

The strategy for the small scale purification of amyloid proteins depends largely on the type of tissue sample (fresh or formalin fixed), sample size, amyloid content of the tissue, and its chemical nature. Aqueous acetonitrile/TFA solvent was effectively used for the microextraction of different amyloid proteins from non-fixed tissues, although it was unsuitable for the extraction of amyloid proteins from formalin fixed tissues, where harsher treatments were needed to achieve amyloid solubilisation. Reversed phase HPLC was used successfully for the purification and analysis of different types of amyloid proteins recovered from formalin fixed tissues. However, in the examination of fresh tissues, purification by HPLC was efficient only for some amyloid proteins, depending on their chemical type and molecular mass. SDS-PAGE based western blotting appears to be a more universal approach for the initial analysis of amyloid proteins microextracted from non-fixed tissues. It allows the type, molecular mass, and amount of amyloid to be determined. Where the amount of amyloid is sufficient, an SDS-PAGE based microsequencing technique could provide information on the primary amyloid protein structure. When the amount of amyloid is small and contamination by other coextracted tissue proteins is high, the combined use of electrophoretic and HPLC techniques seems to be promising.